Genes and uses for plant improvement

ABSTRACT

Transgenic seed for crops with improved traits are provided by trait-improving recombinant DNA where plants grown from such transgenic seed exhibit one or more improved traits as compared to a control plant. Also provided are methods of making transgenic plants with recombinant DNA and the one or more improved traits and methods of using those plants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/121,854, filed Oct. 24, 2014, which application is a continuation ofU.S. application Ser. No. 11/982,700, filed Nov. 1, 2007, whichapplication is a divisional of U.S. application Ser. No. 11/188,298,filed Jul. 22, 2005, which claims the benefit of priority to U.S.Provisional Application No. 60/592,978, filed Jul. 31, 2004, whichapplications are incorporated herein by reference and made a part hereofin their entirety, and the benefit of priority of each of which isclaimed herein.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy 2) and a computerreadable form (CRF) of the sequence listing, all on CD-ROMs, eachcontaining the file named “3126.002US4 seq listing in text-crf(1379958x7ADA8).TXT”, which is 70,686,720 bytes (measured in MS-WINDOWS)and was created on Apr. 5, 2017, are herein incorporated by reference.

FIELD OF THE INVENTION

Disclosed herein are inventions in the field of plant genetics anddevelopmental biology. More specifically, this invention providestransgenic seeds for crops, wherein the genome of said seed comprisesrecombinant DNA, the expression of which results in the production oftransgenic plants that have improved trait(s).

BACKGROUND OF THE INVENTION

Transgenic plants with improved traits, such as improved yield,environmental stress tolerance, pest resistance, herbicide tolerance,modified seed compositions, and the like are desired by both farmers andconsumers. Although considerable efforts in plant breeding have providedsignificant gains in desired traits, the ability to introduce specificDNA into plant genomes provides further opportunities for generation ofplants with improved and/or unique traits. The ability to developtransgenic plants with improved traits depends in part on theidentification of genes that are useful in recombinant DNA constructsfor production of transformed plants with improved properties.

SUMMARY OF THE INVENTION

This invention provides transgenic seeds, transgenic plants and DNAconstructs with trait-improving recombinant DNA from a gene for aprotein having an amino acid sequence with at least 90% identity to aconsensus amino acid sequence in the group consisting of SEQ ID NO: 270and its homologs through SEQ ID NO: 538, where the respective homologproteins have amino acid sequences SEQ ID NO: 539 through SEQ ID NO:22568, as indicated in Table 16. In some cases of trait improvement, therecombinant DNA encodes a protein; in other cases, the recombinant DNAsuppresses endogenous protein expression. In a broad aspect thisinvention provides transgenic seeds for growing crop plants withimproved traits, such crop plants with improved traits and the plantparts including transgenic seed produced by such crop plants. Theimproved traits provided by the recombinant DNA in the transgenic cropplant of this invention are identified by comparison to a control plant,i.e., a plant without the trait-improving recombinant DNA. In one aspectof the invention, transgenic crop plant grown from the transgenic seedhas improved yield, as compared to the yield of a control plant, e.g., aplant without the recombinant DNA that produces the increased yield.Some plants of this invention exhibit increased yield by producing ayield increase under non-stress conditions. Other plants of thisinvention exhibit increased yield by producing a yield increase underone or more environmental stress conditions including, but not limitedto, water deficit stress, cold stress, heat stress, high salinitystress, shade stress, and low nitrogen availability stress. Still otherplants of this invention have other improved phenotypes, such asimproved plant development, plant morphology, plant physiology or seedcomposition as compared to a corresponding trait of a control plant. Thevarious aspects of this invention are especially useful for transgenicseed and transgenic plants having improved traits in corn (maize),soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa,barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

The invention also comprises recombinant DNA constructs. In one aspect,such recombinant DNA constructs useful for the transgenic seed andtransgenic plants of this invention comprise a promoter functional in aplant cell operably linked to a DNA segment for expressing a proteinassociated with a trait in a model plant or a homologue. In anotheraspect the recombinant DNA constructs useful for the transgenic seed andtransgenic plants of this invention comprise a promoter functional in aplant cell operably linked to a DNA segment for suppressing the level ofan endogenous plant protein which is a homologue to a model-plantprotein, the suppression of which is associated with an improved trait.Suppression can be effected by any of a variety of methods known in theart, e.g., post transcriptional suppression by anti-sense, sense, dsRNAand the like or by transcriptional suppression.

This invention also provides a method of producing a transgenic cropplant having at least one improved trait, wherein the method comprisesproviding to a grower of transgenic seeds comprising recombinant DNA forexpression or suppression of a trait-improving gene provided herein, andgrowing transgenic plant from said transgenic seed. Such methods areused to generate transgenic crop plants having at least one improvedtrait under one or more environmental stress conditions including, butnot limited to, water deficit stress, cold stress, heat stress, highsalinity stress, shade stress, and low nitrogen availability stress. Inanother aspect, such methods are used to generate transgenic crop plantshaving improved plant development, plant morphology, plant physiology orseed component phenotype as compared to a corresponding phenotype of acontrol plant. Of particular interest are uses of such methods togenerate transgenic crop plants having increased yield under non-stresscondition, or under one or more stress conditions.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides transgenic plant seed having in its genometrait-improving recombinant DNA and transgenic plants grown from suchseed which exhibit an improved trait as compared a control plant. In oneaspect, the invention provides transgenic plants where the improvedtrait is one or more of improved drought stress tolerance, improved heatstress tolerance, improved cold stress tolerance, improved high salinitystress tolerance, improved low nitrogen availability stress tolerance,improved shade stress tolerance, improved plant growth and developmentat the stages of seed imbibition through early vegetative phase, andimproved plant growth and development at the stages of leaf development,flower production and seed maturity. Particular transgenic plants grownfrom transgenic seeds of this invention exhibit increased seed yield.Recombinant DNA constructs used in this invention comprise recombinantDNA disclosed herein which produces mRNA to modulate gene expressionimparting improved traits to plants.

“Gene” means all or part of the DNA that encodes a protein or mRNA,e.g., chromosomal DNA, plasmid DNA, cDNA, or synthetic DNA, and includesDNA regions flanking the coding sequences, e.g., introns, 5′UTR, 3′ UTR,promoters and other DNA involved in the regulation of expression.

“Transgenic seed” means plant seed having a genome altered by theincorporation of recombinant DNA, e.g., by transformation. “Transgenicplant” means a plant produced from an original transformation event, orprogeny from later generations or crosses of a plant to a transformedplant, so long as the progeny contains the recombinant DNA in itsgenome. “Recombinant DNA” means a DNA molecule having a geneticallyengineered modification introduced through a combination of endogenousand/or exogenous DNA elements in a transcription unit, manipulation viamutagenesis, restriction enzymes, and the like or simply by insertingmultiple copies of a native transcription unit. Recombinant DNA maycomprise DNA segments obtained from different sources, or DNA segmentsobtained from the same source, but which have been manipulated to joinDNA segments which do not naturally exist in the joined form.Recombinant DNA can exist outside of a cell, e.g., as a PCR fragment orin a plasmid, or can be integrated into a genome such as a plant genome.

“Trait” means a physiological, morphological, biochemical, or physicalcharacteristic of a plant or particular plant material or cell. In someinstances the characteristic is visible to the human eye, e.g., seed orplant size, or can be measured by biochemical techniques, e.g.,detecting the protein, starch, or oil content of seed or leaves, or byobservation of a metabolic or physiological process, e.g., by measuringuptake of carbon dioxide, or by the observation of the expression levelof a gene or genes, e.g., by employing Northern analysis, RT-PCR,microarray gene expression assays, or reporter gene expression systems,or by agricultural observations such as stress tolerance, yield, orpathogen tolerance.

“Control plant” is a plant without trait-improving recombinant DNA. Acontrol plant is used to measure and compare trait improvement in atransgenic plant with such trait-improving recombinant DNA. One suitablecontrol plant is a non-transgenic plant of the parental line that wasused to generate a transgenic plant. Another suitable control plant is atransgenic plant that comprises recombinant DNA without the specifictrait producing DNA, e.g., simply a marker gene. Another suitablecontrol plant is a negative segregant progeny of hemizygous transgenicplant. In certain demonstrations of trait improvement, e.g., in fieldconditions, the use of a limited number of control plants can cause awide variation in the control dataset. To minimize the effect of thevariation within the control dataset, a “reference” is used, i.e., atrimmed mean of all data from both transgenic and control plants grownunder the same conditions and at the same developmental stage. Thetrimmed mean is calculated by eliminating a specific percentage, i.e.,20%, of the smallest and largest observation from the data set and thencalculating the average of the remaining observation.

“Trait improvement” means a detectable and desirable difference in acharacteristic in a transgenic plant relative to a control plant or areference. In some cases, the trait improvement is measuredquantitatively. For example, the trait improvement can entail at least a2% desirable difference in an observed trait, at least a 5% desirabledifference, at least about a 10% desirable difference, at least about a20% desirable difference, at least about a 30% desirable difference, atleast about a 50% desirable difference, at least about a 70% desirabledifference, or at least about a 100% difference, or an even greaterdesirable difference. In other cases, the trait improvement is onlymeasured qualitatively. It is known that there are natural variations ina trait. Therefore, the trait improvement observed entails a change ofthe normal distribution of the trait in the transgenic plant comparedwith the trait distribution observed in a control plant or a reference,which is evaluated by statistical methods provided herein. Traitimprovement includes, but not limited to, yield increase, includingincreased yield under non-stress conditions and increased yield underenvironmental stress conditions. Stress conditions may include, forexample, drought, shade, fungal disease, viral disease, bacterialdisease, insect infestation, nematode infestation, cold temperatureexposure, heat exposure, osmotic stress, reduced nitrogen nutrientavailability, reduced phosphorus nutrient availability and high plantdensity. Many agronomic traits can affect “yield”, including withoutlimitation, plant height, pod number, pod position on the plant, numberof internodes, incidence of pod shatter, grain size, efficiency ofnodulation and nitrogen fixation, efficiency of nutrient assimilation,resistance to biotic and abiotic stress, carbon assimilation, plantarchitecture, resistance to lodging, percent seed germination, seedlingvigor, and juvenile traits. Other traits that can affect yield include,efficiency of germination (including germination in stressedconditions), growth rate (including growth rate in stressed conditions),ear number, seed number per ear, seed size, composition of seed (starch,oil, protein) and characteristics of seed fill. Also of interest is thegeneration of transgenic plants that demonstrate desirable phenotypicproperties that may or may not confer an increase in overall plantyield. Such properties include improved plant morphology, plantphysiology or improved components of the mature seed harvested from thetransgenic plant.

“Yield-limiting environment” means a condition under which a plant wouldhave the limitation on yield including environmental stress conditions.

“Stress condition” means a condition unfavorable for a plant, whichadversely affects plant metabolism, growth and/or development. A plantunder the stress condition typically shows reduced germination rate,retarded growth and development, reduced photosynthesis rate, andeventually leading to reduction in yield. Specifically, “water deficitstress” means sub-optimal conditions for water and humidity needed fornormal growth of natural plants. Relative water content (RWC) is onephysiological measure of plant water deficit. RWC measures the effect ofosmotic adjustment in plant water status, when a plant is under stressedconditions. RWC can result from heat, drought, high salinity and inducedosmotic stress.

“Cold stress” means exposure of a plant to temperatures below, e.g., atleast two or more degrees Celsius below, those temperatures that arenormal for a particular species or particular strain of plant.

“Sufficient nitrogen growth condition” means a growth condition wherethe soil or growth medium contains or receives enough amounts ofnitrogen nutrient to sustain a healthy plant growth and/or for a plantto reach its typical yield for a particular plant species or aparticular strain. “Nitrogen nutrient” means any one or any mix of thenitrate salts commonly used as plant nitrogen fertilizer, including, butnot limited to, potassium nitrate, calcium nitrate, sodium nitrate,ammonium nitrate. “Ammonium” means any one or any mix of the ammoniumsalts commonly used as plant nitrogen fertilizer, e.g., ammoniumnitrate, ammonium chloride, ammonium sulfate, etc. Those skilled in theart know what constitutes such soil, media and fertilizer inputs formost plant species. “Low nitrogen availability stress” means a plantgrowth condition that does not contain sufficient nitrogen nutrient tomaintain a healthy plant growth and/or for a plant to reach its typicalyield under a sufficient nitrogen growth condition; a useful lownitrogen availability stress is a growth condition with 50% or less ofthe conventional nitrogen inputs.

“Shade stress” means a limited light availability that triggers theshade avoidance response in plant. Plants are subject to shade stresswhen localized at lower part of the canopy, or in close proximity ofneighboring vegetation. Shade stress is exacerbated when the plantingdensity exceeds the average prevailing density for a particular plantspecies. The average prevailing densities per acre of a few otherexamples of crop plants in the USA in the year 2000 were: wheat1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000,canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000plants per acre.

“Increased yield” of a transgenic plant of this invention is evidencedand measured in a number of ways, including test weight, seed number perplant, seed weight, seed number per unit area (i.e., seeds, or weight ofseeds, per acre), bushels per acre, tons per acre, tons per acre, kiloper hectare. For example, corn yield is measured as production ofshelled corn kernels per unit of production area, e.g., in bushels peracre or metric tons per hectare, often reported on a moisture adjustedbasis, e.g., at 15.5% moisture. Increased yield is often achieved fromimproved utilization of key biochemical compounds, such as nitrogen,phosphorous and carbohydrate, or from improved responses toenvironmental stresses, such as cold, heat, drought, salt, and attack bypests or pathogens. Trait-improving recombinant DNA is used to providetransgenic plants having improved growth and development, and ultimatelyincreased yield, as the result of modified expression of plant growthregulators or modification of cell cycle or photosynthesis pathways.

“Expression” means transcription of DNA to produce RNA. The resultingRNA includes mRNA encoding a protein, antisense RNA that iscomplementary to an mRNA encoding a protein, or an RNA transcriptcomprising a combination of sense and antisense gene regions, such asfor use in RNAi gene suppression. Expression also means production ofencoded protein from mRNA.

“Promoter” means a region of DNA upstream from the start oftranscription and involved in recognition and binding of RNA polymeraseand other proteins to initiate transcription. A “plant promoter” is apromoter capable of initiating transcription in plant cells whether ornot its origin is a plant cell. Exemplary plant promoters include, butare not limited to, those that are obtained from plants, plant viruses,and bacteria which comprise genes expressed in plant cells such asAgrobacterium or Rhizobium. “Tissue preferred” promoters preferentiallyregulate expression in certain tissues, such as leaves, roots, or seeds.“Tissue specific” promoters predominately regulate expression only incertain tissues. “Cell type” specific promoter primarily regulateexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. “Inducible” and “repressible”promoters regulate expression under environmental influences, under theeffect of anaerobic conditions, certain chemicals, or the presence oflight. Tissue specific, tissue preferred, cell type specific, andinducible promoters constitute a class of “non-constitutive” promoters.“Constitutive” promoters are promoters which are active under mostconditions. “Anti-sense orientation” refers to a DNA sequence that isoperably linked to a promoter in an orientation where the anti-sensestrand is transcribed. “Operably linked” refers to an association of twoor more DNA elements in a single construct so that the function of oneis affected by the other. For example, a promoter is operably linkedwith transcribable DNA when it is capable of affecting the expression ofthat DNA; that is, the coding DNA is under the transcriptional controlof the promoter.

“Consensus sequence” means an artificial, amino acid sequence ofconserved parts of the proteins encoded by homologous genes, e.g., asdetermined by a CLUSTALW alignment of amino acid sequence of homologproteins.

“Homologs” means genes that produce functionally similar proteins, e.g.,in the same organism or in different organisms. A gene can be related toa homolog gene by descent from a common ancestral DNA. Homologs includegenes where the relationship is by speciation, e.g., often calledorthologs, or by genetic duplication, e.g., often called paralogs. Morespecifically, “orthologs” include homologs in different species thatevolved from a common ancestral gene by specification. Normallyorthologs retain the same function in the course of evolution.“Paralogs” include homologs in the same species that have diverged fromeach other as a consequence of genetic duplication.

“Percent identity” means the extent to which two optimally aligned DNAor protein segments are invariant throughout a window of alignment ofcomponents, e.g., nucleotide sequence or amino acid sequence. An“identity fraction” for aligned segments of sequences is the number ofidentical components which are shared divided by the total number ofsequence components in the segment used as a reference over a window ofalignment which is the smaller of the sequences. “Percent identity” (“%identity”) is the identity fraction times 100. “% identity” to aconsensus amino acid sequence” is 100 times the identity fraction in awindow of alignment of an amino acid sequence of a test proteinoptimally aligned to consensus amino acid sequence of this invention.

“Arabidopsis” means plants of Arabidopsis thaliana.

Recombinant DNA Constructs

This invention provides recombinant DNA constructs comprising DNAelements for imparting one or more improved traits to transgenic plant.Such constructs typically comprise a promoter operatively linked to DNAto provide for expression of a protein or RNA for gene suppression in atarget plant. Recombinant DNA constructs can also include additionalregulatory elements, such as 5′ or 3′ untranslated regions (UTRs) suchas polyadenylation sites, introns, and transit or signal peptides. Suchrecombinant DNA constructs are assembled using methods known to those ofordinary skill in the art.

In certain embodiments, recombinant DNA constructs comprisesense-oriented, trait-imparting DNA operably linked to a promoter thatis functional in a plant to provide for expression of thetrait-imparting DNA in the sense orientation such that a desired proteinis produced. In other embodiments at least a part of the trait-impartingDNA is in an anti-sense orientation for gene suppression activity.

Recombinant DNA constructs, especially for expressing proteins aretypically prepared with a 3′ UTR that a polyadenylation site and signal.Recombinant DNA constructs can also include a transit peptide fortargeting of a gene target to a plant organelle, particularly to achloroplast, leucoplast or other plastid organelle. For descriptions ofthe use of chloroplast transit peptides, see U.S. Pat. Nos. 5,188,642and 5,728,925, incorporated herein by reference.

Table 1 provides a list of genes that can provide trait-imparting DNAfor recombinant DNA constructs. DNA from each gene was used in a modelplant (Arabidopsis) to discover associations with improved traits. TheDNA was also used to identify homologs from which a consensus amino acidsequence is defined for characterizing the aspects of the inventionwhere recombinant DNA is incorporated in the transgenic seeds,transgenic plants, DNA constructs and methods of this invention. Withreference to Table 1:

“NUC SEQ ID NO” refers to a SEQ ID NO. for particular DNA sequence inthe Sequence Listing.

“PEP SEQ ID NO” refers to a SEQ ID NO. in the Sequence Listing for theamino acid sequence of a protein cognate to a particular DNA

“construct_id” refers to an arbitrary number used to identify aparticular recombinant DNA construct comprising the particular DNA.

“gene” refers to an arbitrary name used to identify the particular DNA.

“orientation” refers to the orientation of the particular DNA in arecombinant DNA construct relative to the promoter.

“species” refers to the organism from which the particular DNA wasderived.

TABLE 1 Nuc SEQ ID Pep SEQ ID construct_id Gene orientation Species 1270 14324 CGPG1560 SENSE Arabidopsis thaliana 2 271 17484 CGPG2630 SENSEArabidopsis thaliana 3 272 19109 CGPG1381 ANTI-SENSE Arabidopsisthaliana 4 273 70423 CGPG3165 SENSE Arabidopsis thaliana 5 274 70424CGPG3180 SENSE Arabidopsis thaliana 6 275 70480 CGPG3833 SENSEArabidopsis thaliana 7 276 70509 CGPG2420 SENSE Arabidopsis thaliana 8277 70647 CGPG4334 SENSE Arabidopsis thaliana 9 278 70675 CGPG4519 SENSEArabidopsis thaliana 10 279 70829 CGPG518 SENSE Arabidopsis thaliana 11280 70849 CGPG596 SENSE Arabidopsis thaliana 12 281 71627 CGPG1270 SENSEArabidopsis thaliana 13 282 71934 CGPG2294 SENSE Arabidopsis thaliana 14283 72615 CGPG4829 SENSE Arabidopsis thaliana 15 284 72927 CGPG1477SENSE Arabidopsis thaliana 16 285 73014 CGPG5692 SENSE Xenorhabdusnematophilus 85816 17 286 73559 CGPG6535 SENSE Bacillus subtilis 168 18287 74251 CGPG5489 SENSE Arabidopsis thaliana 19 288 19631 CGPG3627SENSE Arabidopsis thaliana 20 289 70121 CGPG2380 SENSE Saccharomycescerevisiae 21 290 70654 CGPG4352 SENSE Arabidopsis thaliana 22 291 70696CGPG4590 SENSE Arabidopsis thaliana 23 292 70713 CGPG1462 ANTI-SENSEArabidopsis thaliana 24 293 70740 CGPG3700 SENSE Arabidopsis thaliana 25294 71321 CGPG4418 SENSE Arabidopsis thaliana 26 295 71835 CGPG4634SENSE Arabidopsis thaliana 27 296 72934 CGPG5798 SENSE Saccharomycescerevisiae 28 297 72945 CGPG5787 SENSE Saccharomyces cerevisiae 29 29872980 CGPG5773 SENSE Saccharomyces cerevisiae 30 299 73504 CGPG6480SENSE Synechocystis sp. PCC 6803 31 300 73507 CGPG6504 SENSE Bacillussubtilis 168 32 301 73573 CGPG6462 SENSE Agrobacterium tumefacians C5833 302 73586 CGPG6471 SENSE Bacillus subtilis 168 34 303 73770 CGPG5435SENSE Arabidopsis thaliana 35 304 74105 CGPG6574 SENSE Xenorhabdusnematophilus 86068 36 305 74111 CGPG6622 SENSE Escherichia coli K-12 37306 74136 CGPG6632 SENSE Synechocystis 38 307 74139 CGPG6561 SENSEEscherichia coli K-12 39 308 74267 CGPG5364 SENSE Arabidopsis thaliana40 309 74291 CGPG5363 SENSE Arabidopsis thaliana 41 310 74318 CGPG5826SENSE Arabidopsis thaliana 42 311 74319 CGPG5831 SENSE Arabidopsisthaliana 43 312 74324 CGPG5885 SENSE Arabidopsis thaliana 44 313 74512CGPG32 SENSE Arabidopsis thaliana 45 314 74583 CGPG6649 SENSE Ralstoniametallidurans CH34 46 315 70427 CGPG3067 SENSE Arabidopsis thaliana 47316 71811 CGPG4426 SENSE Arabidopsis thaliana 48 317 73463 CGPG6384SENSE Ralstonia metallidurans CH34 49 318 72081 CGPG5279 SENSE Glycinemax 50 319 10139 CGPG101 ANTI-SENSE Arabidopsis thaliana 51 320 11410CGPG103 SENSE Arabidopsis thaliana 52 321 11604 CGPG48 ANTI-SENSEArabidopsis thaliana 53 322 12368 CGPG1006 SENSE Arabidopsis thaliana 54323 13502 CGPG1354 SENSE Arabidopsis thaliana 55 324 13745 CGPG1576ANTI-SENSE Arabidopsis thaliana 56 325 13821 CGPG1569 SENSE Arabidopsisthaliana 57 326 14240 CGPG1697 SENSE Arabidopsis thaliana 58 327 14718CGPG1082 SENSE Arabidopsis thaliana 59 328 17022 CGPG1774 SENSEArabidopsis thaliana 60 329 17924 CGPG2882 SENSE Arabidopsis thaliana 61330 18259 CGPG3368 SENSE Arabidopsis thaliana 62 331 19171 CGPG2952SENSE Saccharomyces cerevisiae 63 332 19201 CGPG2332 SENSE Arabidopsisthaliana 64 333 19317 CGPG3662 SENSE Xanthomonas 65 334 70417 CGPG3427SENSE Arabidopsis thaliana 66 335 70467 CGPG3785 SENSE Arabidopsisthaliana 67 336 70806 CGPG712 SENSE Arabidopsis thaliana 68 337 70818CGPG479 SENSE Arabidopsis thaliana 69 338 70820 CGPG655 SENSEArabidopsis thaliana 70 339 70919 CGPG4029 SENSE Glycine max 71 34071623 CGPG4696 SENSE Arabidopsis thaliana 72 341 71662 CGPG4679 SENSEGlycine max 73 342 71693 CGPG4652 SENSE Glycine max 74 343 72384CGPG4639 SENSE Saccharomyces cerevisiae 75 344 72439 CGPG5075 SENSEArabidopsis thaliana 76 345 72619 CGPG4835 SENSE Arabidopsis thaliana 77346 72624 CGPG4842 SENSE Arabidopsis thaliana 78 347 72715 CGPG5521SENSE Saccharomyces cerevisiae 79 348 72754 CGPG5548 SENSE Saccharomycescerevisiae 80 349 72819 CGPG4989 SENSE Arabidopsis thaliana 81 350 75516CGPG7689 SENSE Glycine max 82 351 75701 CGPG7856 SENSE Glycine max 83352 73515 CGPG6473 SENSE Bacillus subtilis 168 84 353 74684 CGPG6360SENSE Arabidopsis thaliana 85 354 19542 CGPG3069 SENSE Arabidopsisthaliana 86 355 19618 CGPG3574 SENSE Arabidopsis thaliana 87 356 19649CGPG3140 SENSE Arabidopsis thaliana 88 357 19745 CGPG3973 SENSE Glycinemax 89 358 19768 CGPG4096 SENSE Glycine max 90 359 19772 CGPG3939 SENSEGlycine max 91 360 19779 CGPG4113 SENSE Glycine max 92 361 19833CGPG4074 SENSE Glycine max 93 362 19862 CGPG3961 SENSE Glycine max 94363 19879 CGPG4009 SENSE Glycine max 95 364 70445 CGPG3728 SENSEArabidopsis thaliana 96 365 70738 CGPG3195 SENSE Arabidopsis thaliana 97366 71437 CGPG4043 SENSE Glycine max 98 367 71572 CGPG4520 SENSEArabidopsis thaliana 99 368 71617 CGPG1227 SENSE Arabidopsis thaliana100 369 72532 CGPG4780 SENSE Arabidopsis thaliana 101 370 72757 CGPG5572SENSE Arabidopsis thaliana 102 371 73412 CGPG6448 SENSE Pseudomonassyringae var tomato DC3000 103 372 74102 CGPG6550 SENSE Bacillushalodurans C-125 104 373 72633 CGPG4853 SENSE Arabidopsis thaliana 105374 72456 CGPG4745 SENSE Arabidopsis thaliana 106 375 72963 CGPG1746SENSE Arabidopsis thaliana 107 376 70426 CGPG3199 SENSE Arabidopsisthaliana 108 377 70772 CGPG4627 SENSE Arabidopsis thaliana 109 378 71137CGPG125 SENSE Arabidopsis thaliana 110 379 71529 CGPG2808 SENSEArabidopsis thaliana 111 380 71601 CGPG1858 SENSE Arabidopsis thaliana112 381 72362 CGPG983 SENSE Arabidopsis thaliana 113 382 72466 CGPG4767SENSE Arabidopsis thaliana 114 383 72524 CGPG4770 SENSE Arabidopsisthaliana 115 384 73085 CGPG5689 SENSE Synechocystis sp. PCC 6803 116 38574241 CGPG5457 SENSE Arabidopsis thaliana 117 386 74247 CGPG5475 SENSEArabidopsis thaliana 118 387 74284 CGPG5413 SENSE Arabidopsis thaliana119 388 74652 CGPG6168 SENSE Arabidopsis thaliana 120 389 70437 CGPG3706SENSE Arabidopsis thaliana 121 390 71633 CGPG857 SENSE Arabidopsisthaliana 122 391 72948 CGPG5617 SENSE Arabidopsis thaliana 123 392 72519CGPG4749 SENSE Arabidopsis thaliana 124 393 10475 CGPG399 SENSEArabidopsis thaliana 125 394 11120 CGPG459 ANTI-SENSE Arabidopsisthaliana 126 395 19736 CGPG4129 SENSE Glycine max 127 396 71606 CGPG4715SENSE Arabidopsis thaliana 128 397 71840 CGPG4353 SENSE Arabidopsisthaliana 129 398 74240 CGPG5454 SENSE Arabidopsis thaliana 130 399 74331CGPG5834 SENSE Arabidopsis thaliana 131 400 74610 CGPG6048 SENSEArabidopsis thaliana 132 401 75527 CGPG7682 SENSE Glycine max 133 40270681 CGPG4584 SENSE Arabidopsis thaliana 134 403 71663 CGPG4638 SENSEXanthomonas 135 404 72769 CGPG5573 SENSE Arabidopsis thaliana 136 40571508 CGPG1541 SENSE Arabidopsis thaliana 137 406 74248 CGPG5476 SENSEArabidopsis thaliana 138 407 72771 CGPG2166 SENSE Arabidopsis thaliana139 408 72085 CGPG5228 SENSE Arabidopsis thaliana 140 409 72744 CGPG5563SENSE Saccharomyces cerevisiae 141 410 73039 CGPG810 SENSE Arabidopsisthaliana 142 411 73054 CGPG5754 SENSE Saccharomyces cerevisiae 143 41273501 CGPG6456 SENSE Agrobacterium tumefacians C58 144 413 19707CGPG4179 SENSE Glycine max 145 414 19951 CGPG3941 SENSE Glycine max 146415 19967 CGPG4032 SENSE Glycine max 147 416 70543 CGPG3815 SENSEArabidopsis thaliana 148 417 70707 CGPG1273 ANTI-SENSE Arabidopsisthaliana 149 418 70719 CGPG1712 ANTI-SENSE Arabidopsis thaliana 150 41971134 CGPG817 SENSE Arabidopsis thaliana 151 420 71146 CGPG2928 SENSEArabidopsis thaliana 152 421 71660 CGPG4690 SENSE Arabidopsis thaliana153 422 72086 CGPG5236 SENSE Arabidopsis thaliana 154 423 72632 CGPG4852SENSE Arabidopsis thaliana 155 424 72716 CGPG5529 SENSE Saccharomycescerevisiae 156 425 72723 CGPG1848 SENSE Arabidopsis thaliana 157 42672987 CGPG1787 SENSE Arabidopsis thaliana 158 427 74109 CGPG6606 SENSEXenorhabdus nematophilus 86068 159 428 74140 CGPG6569 SENSE Bacillushalodurans C-125 160 429 74191 CGPG6597 SENSE Rhodobacter sphaeroides2.4.1 161 430 74265 CGPG5356 SENSE Arabidopsis thaliana 162 431 74369CGPG6076 SENSE Arabidopsis thaliana 163 432 70217 CGPG6 SENSEArabidopsis thaliana 164 433 72711 CGPG1846 SENSE Arabidopsis thaliana165 434 70932 CGPG4089 SENSE Glycine max 166 435 73518 CGPG6497 SENSEPseudomonas fluorescens PfO-1 167 436 19771 CGPG4011 SENSE Glycine max168 437 73549 CGPG6460 SENSE Xenorhabdus nematophilus 85816 169 43872994 CGPG5803 SENSE Saccharomyces cerevisiae 170 439 71928 CGPG1617SENSE Arabidopsis thaliana 171 440 72903 CGPG5584 SENSE Arabidopsisthaliana 172 441 73017 CGPG5733 SENSE Saccharomyces cerevisiae 173 44274587 CGPG6774 SENSE Agrobacterium tumefacians C58 174 443 72453CGPG4735 SENSE Arabidopsis thaliana 175 444 72967 CGPG5742 SENSESaccharomyces cerevisiae 176 445 72961 CGPG5591 SENSE Arabidopsisthaliana 177 446 73070 CGPG5627 SENSE Arabidopsis thaliana 178 447 73475CGPG6385 SENSE Rhodopseudomonas palustris CGA009 179 448 72916 CGPG1814SENSE Arabidopsis thaliana 180 449 72969 CGPG5789 SENSE Saccharomycescerevisiae 181 450 74449 CGPG6659 SENSE Agrobacterium tumefaciens 182451 16615 CGPG2539 SENSE Agrobacterium 183 452 19187 CGPG3310 SENSEArabidopsis thaliana 184 453 19648 CGPG3134 SENSE Arabidopsis thaliana185 454 70354 CGPG3995 SENSE Glycine max 186 455 70421 CGPG2942 SENSEArabidopsis thaliana 187 456 70459 CGPG3758 SENSE Arabidopsis thaliana188 457 70465 CGPG3775 SENSE Arabidopsis thaliana 189 458 70683 CGPG4587SENSE Arabidopsis thaliana 190 459 70725 CGPG2097 ANTI-SENSE Arabidopsisthaliana 191 460 70852 CGPG1465 SENSE Arabidopsis thaliana 192 461 71112CGPG934 SENSE Arabidopsis thaliana 193 462 71127 CGPG945 SENSEArabidopsis thaliana 194 463 71132 CGPG1561 SENSE Arabidopsis thaliana195 464 71217 CGPG95 SENSE Arabidopsis thaliana 196 465 71645 CGPG4688SENSE Arabidopsis thaliana 197 466 71726 CGPG3894 SENSE Arabidopsisthaliana 198 467 72432 CGPG4562 SENSE Arabidopsis thaliana 199 468 72450CGPG4732 SENSE Arabidopsis thaliana 200 469 72455 CGPG4742 SENSEArabidopsis thaliana 201 470 72727 CGPG5522 SENSE Saccharomycescerevisiae 202 471 72817 CGPG4987 SENSE Arabidopsis thaliana 203 47272992 CGPG5777 SENSE Saccharomyces cerevisiae 204 473 73007 CGPG5760SENSE Saccharomyces cerevisiae 205 474 73073 CGPG5688 SENSESynechocystis sp. PCC 6803 206 475 73506 CGPG6496 SENSE Pseudomonasfluorescens PfO-1 207 476 74107 CGPG6590 SENSE Sinorhizobium meliloti1021 208 477 74117 CGPG6575 SENSE Xenorhabdus nematophilus 86068 209 47874131 CGPG6592 SENSE Synechocystis sp. PCC 6803 210 479 74344 CGPG5929SENSE Arabidopsis thaliana 211 480 14320 CGPG1229 SENSE Arabidopsisthaliana 212 481 16756 CGPG2117 SENSE Arabidopsis thaliana 213 482 17448CGPG2673 SENSE Arabidopsis thaliana 214 483 17633 CGPG2839 SENSEArabidopsis thaliana 215 484 18876 CGPG3096 SENSE Arabidopsis thaliana216 485 19120 CGPG1976 ANTI-SENSE Arabidopsis thaliana 217 486 19221CGPG2958 SENSE Arabidopsis thaliana 218 487 70206 CGPG4116 SENSE Glycinemax 219 488 70223 CGPG53 SENSE Arabidopsis thaliana 220 489 70347CGPG3147 SENSE Arabidopsis thaliana 221 490 70406 CGPG1687 SENSEArabidopsis thaliana 222 491 70469 CGPG3791 SENSE Arabidopsis thaliana223 492 70564 CGPG1864 SENSE Arabidopsis thaliana 224 493 70601 CGPG2917SENSE Arabidopsis thaliana 225 494 70612 CGPG3721 SENSE Arabidopsisthaliana 226 495 70720 CGPG1358 ANTI-SENSE Arabidopsis thaliana 227 49670735 CGPG2661 SENSE Arabidopsis thaliana 228 497 70846 CGPG377 SENSEArabidopsis thaliana 229 498 70923 CGPG4020 SENSE Glycine max 230 49971149 CGPG3457 SENSE Arabidopsis thaliana 231 500 71608 CGPG4687 SENSEArabidopsis thaliana 232 501 71739 CGPG4345 SENSE Arabidopsis thaliana233 502 72014 CGPG5230 SENSE Arabidopsis thaliana 234 503 72051 CGPG5241SENSE Arabidopsis thaliana 235 504 74259 CGPG5343 SENSE Arabidopsisthaliana 236 505 72463 CGPG4760 SENSE Arabidopsis thaliana 237 506 72902CGPG5597 SENSE Arabidopsis thaliana 238 507 74572 CGPG6640 SENSESynechocystis 239 508 73055 CGPG5768 SENSE Saccharomyces cerevisiae 240509 74103 CGPG6558 SENSE Escherichia coli K-12 241 510 72921 CGPG5781SENSE Saccharomyces cerevisiae 242 511 72968 CGPG5772 SENSESaccharomyces cerevisiae 243 512 19703 CGPG4172 SENSE Glycine max 244513 19946 CGPG4097 SENSE Glycine max 245 514 19980 CGPG3914 SENSEGlycine max 246 515 70435 CGPG3701 SENSE Arabidopsis thaliana 247 51671114 CGPG1657 SENSE Arabidopsis thaliana 248 517 72451 CGPG4733 SENSEArabidopsis thaliana 249 518 72947 CGPG5607 SENSE Glycine max 250 51973012 CGPG5786 SENSE Saccharomyces cerevisiae 251 520 73022 CGPG5622SENSE Arabidopsis thaliana 252 521 73488 CGPG6394 SENSE Bacillussubtilis 168 253 522 73901 CGPG5237 SENSE Arabidopsis thaliana 254 52373964 CGPG5804 SENSE Saccharomyces cerevisiae 255 524 74019 CGPG5706SENSE Bacillus subtilis 168 256 525 74022 CGPG5724 SENSE Arabidopsisthaliana 257 526 74114 CGPG6551 SENSE Agrobacterium tumefacians C58 258527 74262 CGPG5353 SENSE Arabidopsis thaliana 259 528 74292 CGPG5367SENSE Arabidopsis thaliana 260 529 74302 CGPG5384 SENSE Arabidopsisthaliana 261 530 74325 CGPG5898 SENSE Arabidopsis thaliana 262 531 74429CGPG6689 SENSE Bacillus subtilis 168 263 532 74440 CGPG6682 SENSEBacillus halodurans C-125 264 533 74462 CGPG6668 SENSE Synechocystis 265534 74465 CGPG6692 SENSE Bacillus subtilis 168 266 535 74474 CGPG6669SENSE Synechocystis 267 536 74505 CGPG6783 SENSE Escherichia coli K-12268 537 74507 CGPG6799 SENSE Xenorhabdus nematophilus 85816 269 53874562 CGPG6764 SENSE Bacillus subtilis 168

Recombinant DNA

Trait-imparting DNA for use in this invention for improved traits inplants is disclosed herein as having a DNA sequence of SEQ ID NO:1through SEQ ID NO:269 and any of the respective homologs. A subset ofthe trait-imparting DNA includes fragments with less than the full DNAsequence, e.g., consisting of oligonucleotides of at least about 15 to20 or more consecutive nucleotides from one of the disclosed sequences.Such oligonucleotides are fragments of the larger molecules having asequence selected from the group consisting of SEQ ID NO: 1 through SEQID NO: 269, and find use, for example as probes and primers fordetection of the polynucleotides of the invention or for cloning DNA foruse in this invention.

Useful DNA includes variants of the disclosed DNA. Such variants includenaturally occurring, including homologous DNA from genes of the same ora different species, or non-natural variants, for example DNAsynthesized using chemical synthesis methods, or generated usingrecombinant DNA techniques. Degeneracy of the genetic code provides thepossibility to substitute at least one nucleotide of a disclosed DNAwithout causing the amino acid sequence of the protein produced to bechanged. Hence, useful DNA can have any base sequence that has beenchanged from the sequences provided herein by substitution in accordancewith degeneracy of the genetic code.

Homologs of the trait-imparting DNA generally demonstrate significantidentity with the DNA provided herein. Homologous DNA is substantiallyidentical to a trait-imparting DNA if, when the nucleotide sequences areoptimally aligned there is at least about 60% nucleotide identity, orhigher, e.g., at least 70% or 80% or 85% or even 90% identity or higher,such as 95% or 98% identity over a comparison window of at least 50 to100 nucleotides, and up to the entire length of the trait-imparting DNA.Optimal alignment of sequences for aligning a comparison window can beconducted by algorithms including computerized implementations of thealgorithms (for example, the Wisconsin Genetics Software Package Release7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). Thereference DNA sequence can represent a full-length coding sequence or aportion.

Proteins useful for imparting improved traits are entire proteins or atleast a sufficient portion of the entire protein to impart the relevantbiological activity of the protein. Proteins useful for generation oftransgenic plants having improved traits include the proteins with anamino acid sequence provided herein as SEQ ID NO: 270 through SEQ ID NO:538, as well as homologs of such proteins.

One method to identify homologs of the proteins useful in this inventionis by comparison of the amino acid sequence of the trait-impartingprotein to amino acid sequences of proteins from the same or differentorganisms, e.g., manually or by using known homology-based searchalgorithms such as those commonly known and referred to as BLAST, FASTA,and Smith-Waterman. In one method a local sequence alignment program,e.g., BLAST, is used to search a database of sequences to find similarsequences, and the summary Expectation value (E-value) is used tomeasure the sequence base similarity. As a protein hit with the bestE-value for a particular organism may not necessarily be an ortholog orthe only ortholog, a reciprocal BLAST search is used to filter hitsequences with significant E-values for ortholog identification. Thereciprocal BLAST entails search of the significant hits against adatabase of amino acid sequences from the base organism that are similarto the sequence of the query protein. A hit is a likely ortholog, whenthe reciprocal BLAST's best hit is the query protein itself or a proteinencoded by a duplicated gene after speciation. Thus, homolog is usedherein to described proteins that are assumed to have functionalsimilarity by inference from sequence base similarity. The relationshipof homologs with amino acid sequences of SEQ ID NO: 539 through SEQ IDNO: 22568 to the proteins with amino acid sequences of SEQ ID NO: 270through SEQ ID NO: 538 is found in Table 16.

Aspects of the invention also use DNA encoding functional homologproteins which differ in one or more amino acids from those of proteinencoded by disclosed trait-imparting DNA as the result of one or more ofthe well-known conservative amino acid substitutions, e.g., valine is aconservative substitute for alanine and threonine is a conservativesubstitute for serine. Conservative substitutions for an amino acidwithin the native sequence are selected from other members of a class towhich the naturally occurring amino acid belongs. Representative aminoacids within these various classes include, but are not limited to: (1)acidic (negatively charged) amino acids such as aspartic acid andglutamic acid; (2) basic (positively charged) amino acids such asarginine, histidine, and lysine; (3) neutral polar amino acids such asglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine; and (4) neutral nonpolar (hydrophobic) amino acids such asalanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine. Conserved substitutes for an amino acidwithin a native amino acid sequence are selected from other members ofthe group to which the naturally occurring amino acid belongs. Forexample, a group of amino acids having aliphatic side chains is glycine,alanine, valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Naturally conservative amino acids substitution groups are:valine-leucine, valine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, aspartic acid-glutamic acid, andasparagine-glutamine. A further aspect of the invention uses DNAencoding proteins that differ in one or more amino acids from those ofprotein encoded from a described trait-imparting DNA as the result ofdeletion or insertion of one or more amino acids in a native sequence.

Homologs of the proteins encoded by disclosed trait-improving DNA willgenerally demonstrate significant sequence identity, e.g., at least 50%amino acid sequence identity or higher such as at least 70% identity orat least 80% or at least 90% identity with an amino acid sequence of SEQID NO:270 through SEQ ID NO:538. Identity of protein homologs isdetermined by optimally aligning the amino acid sequence of a putativeprotein homolog with a defined amino acid sequence of a protein encodedby a disclosed trait-imparting DNA and by calculating the percentage ofidentical and conservatively substituted amino acids over the window ofcomparison. The window of comparison for determining identity can be theentire amino acid sequence disclosed herein, e.g., the full sequence ofany of SEQ ID NO:270 through SEQ ID NO:538.

Genes that are homologs to each other can be grouped into families andincluded in multiple sequence alignments to allow a consensus sequenceto be derived. This analysis enables the derivation of conserved andclass-(family) specific residues or motifs that are functionallyimportant. These conserved residues and motifs can be further validatedwith 3D protein structure if available. A consensus sequence is used todefine the full scope of the invention, e.g., to identify proteins witha homolog relationship and the corresponding trait-imparting DNA. Thus,this invention contemplates that protein homologs include proteins withan amino acid sequence that has at least 90% identity to such aconsensus amino acid sequence.

Promoters

Numerous promoters that are active in plant cells have been described inthe literature. These include promoters present in plant genomes as wellas promoters from other sources, including nopaline synthase (NOS)promoter and octopine synthase (OCS) promoters carried on tumor-inducingplasmids of Agrobacterium tumefaciens, caulimovirus promoters such asthe cauliflower mosaic virus or figwort mosaic virus promoters. Forinstance, see U.S. Pat. Nos. 5,858,742 and 5,322,938 which discloseversions of the constitutive promoter derived from cauliflower mosaicvirus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a FigwortMosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which disclosesa maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a riceactin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter,U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat.No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat.No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No.6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat.No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No.6,084,089 which discloses cold inducible promoters, U.S. Pat. No.6,294,714 which discloses light inducible promoters, U.S. Pat. No.6,140,078 which discloses salt inducible promoters, U.S. Pat. No.6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No.6,175,060 which discloses phosphorus deficiency inducible promoters,U.S. Patent Application Publication 2002/0192813A1 which discloses 5′,3′ and intron elements useful in the design of effective plantexpression vectors, U.S. patent application Ser. No. 09/078,972 whichdiscloses a coixin promoter, U.S. patent application Ser. No. 09/757,089which discloses a maize chloroplast aldolase promoter, and U.S. patentapplication Ser. No. 10/739,565 which discloses water-deficit induciblepromoters, all of which are incorporated herein by reference. These andnumerous other promoters that function in plant cells are known to thoseskilled in the art and available for use in recombinant DNA to providefor expression of desired genes in transgenic plant cells.

It is well known in the art that promoters are usefully altered tocontain multiple “enhancer sequences” to assist in elevating geneexpression. By including an enhancer sequence with such constructs,expression is generally enhanced. These enhancers often are found 5′ tothe start of transcription in a promoter that functions in eukaryoticcells, and can also be inserted in the forward or reverse orientation 5′or 3′ to the coding sequence. In some instances, 5′ enhancing elementsare introns. Particularly useful enhancers are the 5′ introns of therice actin 1 gene and the rice actin 2 gene. Other enhancers includeelements from the CaMV 35S promoter, octopine synthase genes, the maizealcohol dehydrogenase gene, the maize shrunken 1 gene and promoters fromnon-plant eukaryotes.

In some aspects of the invention it is preferred that the promoterelement in the DNA construct be capable of causing sufficient expressionin water deficit conditions. Such promoters can be identified andisolated from the regulatory region of plant genes that are overexpressed in water deficit conditions. Specific water-deficit-induciblepromoters for use in this invention are derived from the 5′ regulatoryregion of genes identified as a heat shock protein 17.5 gene (HSP17.5),an HVA22 gene (HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase(CA4H) gene (CA4H) of Zea maize. Such water-deficit-inducible promotersare disclosed in U.S. 2004-0123347 A1, incorporated herein by reference.

In other aspects of the invention, sufficient expression in plant seedtissues is desired to effect improvements in seed composition. Exemplarypromoters for use for seed composition modification include promotersfrom seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell, et al., (1997)Transgenic Res. 6(2):157-166), globulin 1 (Belanger, et al., (1991)Genetics 129:863-872), glutelin 1 (Russell (1997) supra), andperoxiredoxin antioxidant (Per1) (Stacy, et al., (1996) Plant Mol Biol.31(6):1205-1216).

In still other aspects of the invention, preferential expression inplant green tissues is desired. Promoters of interest for such usesinclude those from genes such as SSU (Fischhoff, et al., (1992) PlantMol Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase(PPDK) (Taniguchi, et al., (2000) Plant Cell Physiol. 41(1):42-48).

Gene Overexpression

“Gene overexpression” means expression, e.g., of a gene at a level inits native host that exceeds levels of expression in a non-transgenichost. In many embodiments of the invention, a recombinant DNA constructprovides gene overexpression, e.g., as identified in Table 1.

Gene Suppression

Gene suppression includes any of the well-known methods for suppressingexpression, typically indicated by reduced levels of protein.Posttranscriptional gene suppression is mediated by transcription ofintegrated recombinant DNA to form double-stranded RNA (dsRNA) havinghomology to a gene targeted for suppression. This formation of dsRNAmost commonly results from transcription of an integrated invertedrepeat of an element of a target gene, and is a common feature of genesuppression methods known as anti-sense suppression, co-suppression andRNA interference (RNAi). Transcriptional suppression can be mediated bya transcribed dsRNA having homology to a promoter DNA sequence to effectwhat is called promoter trans suppression.

More particularly, posttranscriptional gene suppression by inserting arecombinant DNA construct with one or more copies of anti-sense orientedDNA to regulate gene expression in plant cells is disclosed in U.S. Pat.No. 5,107,065 (Shewmaker, et al.,) and U.S. Pat. No. 5,759,829(Shewmaker, et Transgenic plants transformed using such anti-senseoriented DNA constructs for gene suppression can comprise integrated DNAarranged as an inverted repeats that result from insertion of the DNAconstruct into plants by Agrobacterium-mediated transformation, asdisclosed by Redenbaugh, et al., in “Safety Assessment of GeneticallyEngineered Flavr Savr™ Tomato, CRC Press, Inc. (1992). Inverted repeatinsertions can comprises a part or all of the T-DNA construct, e.g., aninverted repeat of a complete transcription unit or an inverted repeatof transcription terminator sequence. Screening for inserted DNAcomprising inverted repeat elements can improve the efficiency ofidentifying transformation events effective for gene silencing whetherthe transformation construct is a simple anti-sense DNA construct whichmust be inserted in multiple copies or a complex inverted repeat DNAconstruct (e.g., an RNAi construct) which can be inserted as a singlecopy.

Posttranscriptional gene suppression by inserting a recombinant DNAconstruct with sense-oriented DNA to regulate gene expression in plantsis disclosed in U.S. Pat. No. 5,283,184 (Jorgensen, et al.) and U.S.Pat. No. 5,231,020 (Jorgensen, et al.). Inserted T-DNA providing genesuppression in plants transformed with such sense constructs byAgrobacterium is organized predominately in inverted repeat structures,as disclosed by Jorgensen, et al., Mol. Gen. Genet., 207:471-477 (1987).See also Stam, et al., The Plant Journal, 12(1), 63-82 (1997) who usedsegregation studies to support Jorgensen's finding that gene silencingis mediated by multimeric transgene T-DNA loci in which the T-DNAs arearranged in inverted repeats. Screening for inserted DNA comprisinginverted repeat elements can improve the gene silencing efficiency whentransforming with simple sense-orientated DNA constructs. Gene silencingefficiency can also be improved by screening for single insertion eventswhen transforming with an RNAi construct containing inverted repeatelements

As disclosed by Redenbaugh, et al., gene suppression can be achieved byinserting into a plant genome recombinant DNA that transcribes dsRNA.Such a DNA insert can be transcribed to an RNA element having the 3′region as a double stranded RNA. RNAi constructs are also disclosed inEP 0426195 A1 (Goldbach, et al., —1991) where recombinant DNA constructsfor transcription into hairpin dsRNA for providing transgenic plantswith resistance to tobacco spotted wilt virus. Double-stranded RNAs werealso disclosed in WO 94/01550 (Agrawal, et al.,) where anti-sense RNAwas stabilized with a self-complementary 3′ segment. Agrawal, et al.,referred to U.S. Pat. No. 5,107,065 for using such self-stabilizedanti-sense RNAs for regulating gene expression in plant cells; seeInternational Publication No. 94/01550. Other double-strandedhairpin-forming elements in transcribed RNA are disclosed inInternational Publication No. 98/05770 (Werner, et al.,) where theanti-sense RNA is stabilized by hairpin forming repeats of poly(CG)nucleotides. See also U.S. Patent Application Publication No.2003/0175965 A1 (Lowe, et al.,) which discloses gene suppression usingand RNAi construct comprising a gene coding sequence preceded byinverted repeats of 5′UTR. See also U.S. Patent Application PublicationNo. 2002/0048814 A1 (Oeller) where RNAi constructs are transcribed tosense or anti-sense RNA which is stabilized by a poly(T)-poly(A) tail.See also U.S. Patent Application Publication No. 2003/0018993 A1(Gutterson, et al.,) where sense or anti-sense RNA is stabilized by aninverted repeat of the 3′ untranslated region of the NOS gene. See alsoU.S. Patent Application Publication No. 2003/0036197 A1 (Glassman, etal.,) where RNA having homology to a target is stabilized by twocomplementary RNA regions.

Gene silencing can also be affected by transcribing RNA from both asense and an anti-sense oriented DNA, e.g., as disclosed by Shewmaker,et al., in U.S. Pat. No. 5,107,065 where in Example 1 a binary vectorwas prepared with both sense and anti-sense aroA genes. See also U.S.Pat. No. 6,326,193 where gene targeted DNA is operably linked toopposing promoters.

Gene silencing can also be affected by transcribing from contiguoussense and anti-sense DNA. In this regard see Sijen, et al., The PlantCell, Vol. 8, 2277-2294 (1996) discloses the use of constructs carryinginverted repeats of a cowpea mosaic virus gene in transgenic plants tomediate virus resistance. Such constructs for posttranscriptional genesuppression in plants by double-stranded RNA are also disclosed inInternational Publication No. WO 99/53050 (Waterhouse, et al,),International Publication No. WO 99/49029 (Graham, et al.), U.S.2004-0029283 A1 (Fillatti), U.S. Pat. No. 6,506,559 (Fire, et al,). Seealso U.S. 2004-0006792 A1 (Shewmaker, et al.,) that discloses constructsand methods for simultaneously expressing one or more recombinant geneswhile simultaneously suppressing one or more native genes in atransgenic plant. See also U.S. Pat. No. 6,448,473 (Mitsky, et al.,)that discloses multi-gene suppression vectors for use in plants. All ofthe above-described patents, applications and international publicationsdisclosing materials and methods for posttranscriptional genesuppression in plants are incorporated herein by reference.Transcriptional suppression such as promoter trans suppression can beaffected by a expressing a DNA construct comprising a promoter operablylinked to inverted repeats of promoter DNA for a target gene. Constructsuseful for such gene suppression mediated by promoter trans suppressionare disclosed by Mette, et al., The EMBO Journal, Vol. 18, No. 1, pp.241-148, 1999 and by Mette, et al., The EMBO Journal, Vol. 19, No. 19,pp. 5194-5201-148, 2000, both of which are incorporated herein byreference.

Suppression can also be achieved by insertion mutations created bytransposable elements may also prevent gene function. For example, inmany dicot plants, transformations with the T-DNA of Agrobacterium arereadily achieved and large numbers of transformants can be rapidlyobtained. Also, some species have lines with active transposableelements that are efficiently be used for the generation of largenumbers of insertion mutations, while some other species lack suchoptions. Mutant plants produced by Agrobacterium or transposonmutagenesis and having altered expression of a polypeptide of interestare identified using the polynucleotides of this invention. For example,a large population of mutated plants are screened to detect mutatedplants having an insertion in the gene encoding the polypeptide ofinterest.

Gene Stacking

This invention also contemplates that the trait-improving recombinantDNA is used in combination with other recombinant DNA to create plantswith a multiple desired traits. The combinations generated includemultiple copies of any one or more of the recombinant DNA constructs.These stacked combinations are created by any method, including but notlimited to cross breeding of transgenic plants, or multiple genetictransformation.

Plant Transformation Methods

Numerous methods for transforming plant cells with recombinant DNA areknown in the art and are useful in producing the transgenic seeds ofthis invention. Two commonly used methods for plant transformation areAgrobacterium-mediated transformation and microprojectile bombardment.Microprojectile bombardment methods are illustrated in U.S. Pat. No.5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No.5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No.6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No.6,153,812 (wheat) and Agrobacterium-mediated transformation is describedin U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean);U.S. Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean),all of which are incorporated herein by reference. For Agrobacteriumtumefaciens based plant transformation system, additional elementspresent on transformation constructs include T-DNA left and right bordersequences to facilitate incorporation of the recombinant polynucleotideinto the plant genome.

In general it is preferred to introduce heterologous DNA randomly, i.e.,at a non-specific location, in the genome of a target plant line. Inspecial cases it is useful to target heterologous DNA insertion in orderto achieve site-specific integration, e.g., to replace an existing genein the genome, to use an existing promoter in the plant genome, or toinsert a recombinant polynucleotide at a predetermined site known to beactive for gene expression. Several site specific recombination systemsexist which are known to function implants include cre-lox as disclosedin U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No.5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced intissue culture on media and in a controlled environment. “Media” meansany of the numerous nutrient mixtures that are used to grow cells invitro, that is, outside of the intact living organism. Recipient celltargets include, but are not limited to, meristem cells, callus,immature embryos and gametic cells such as microspores, pollen, spermand egg cells. It is contemplated that any cell from which a fertileplant is regenerated is useful as a recipient cell. Callus is initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation. Practical transformation methods and materials formaking transgenic plants of this invention, e.g., various media andrecipient target cells, transformation of immature embryos andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. 2004-0216189 A1, whichare incorporated herein by reference.

In practice DNA is introduced into only a small percentage of targetcells in any one experiment. Marker genes are used to provide anefficient system for identification of those cells that are stablytransformed by receiving and integrating a transgenic DNA construct intotheir genomes. Preferred marker genes provide selective markers thatconfer resistance to a selective agent, such as an antibiotic orherbicide. Potentially transformed cells are exposed to the selectiveagent. In the population of surviving cells are those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells are testedfurther to confirm stable integration of the exogenous DNA. Usefulselective marker genes include those conferring resistance toantibiotics such as kanamycin (nptII), hygromycin B (aph IV) andgentamycin (aac3 and aacC4) or resistance to herbicides such asglufosinate (bar or pat) and glyphosate (EPSPS). Examples of suchselectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435;5,780,708 and 6,118,047, all of which are incorporated herein byreference. Screenable markers which provide an ability to visuallyidentify transformants are also often employed, e.g., a gene expressinga colored or fluorescent protein such as a luciferase or greenfluorescent protein (GFP) or a gene expressing a beta-glucuronidase oruidA gene (GUS) for which various chromogenic substrates are known. Itis also contemplated that combinations of screenable and selectablemarkers will be useful for identification of transformed cells. See PCTpublication WO 99/61129 which discloses use of a gene fusion between aselectable marker gene and a screenable marker gene, e.g., an NPTII geneand a GFP gene.

Cells that survive exposure to the selective agent, or cells that havebeen scored positive in a screening assay, are cultured in regenerationmedia and allowed to mature into plants. Developing plantlets aretransferred to soil less plant growth mix, and hardened off, e.g., in anenvironmentally controlled chamber at about 85% relative humidity, 600ppm CO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light, prior to transferto a greenhouse or growth chamber for maturation. Plants are preferablymatured either in a growth chamber or greenhouse. Plants are regeneratedfrom about 6 wk to 10 months after a transformant is identified,depending on the initial tissue. During regeneration, cells are grown toplants on solid media at about 19 to 28 degrees C. After regeneratingplants have reached the stage of shoot and root development, they aretransferred to a greenhouse for further growth and testing. Plants arepollinated using conventional plant breeding methods known to those ofskill in the art and seed produced.

Progeny are recovered from transformed plants and tested for expressionof the exogenous recombinant polynucleotide. Useful assays include, forexample, “molecular biological” assays, such as Southern and Northernblotting and PCR; “biochemical” assays, such as detecting the presenceof RNA, e.g., double stranded RNA, or a protein product, e.g., byimmunological means (ELISAs and Western blots) or by enzymatic function;plant part assays, such as leaf or root assays; and also, by analyzingthe phenotype of the whole regenerated plant.

Discovery of Trait-Improving Recombinant DNA

To identify recombinant DNA that confer improved traits to plants,Arabidopsis plants were transformed with a large population ofrecombinant DNA constructs for expressing a large variety of distinctDNA. Transgenic plants were produced and screened to identify thoseplants having recombinant DNA constructs expressing trait-imparting DNA.A two-step screening process was employed which comprised two passes oftrait characterization to ensure that the trait modification wasdependent on expression of the recombinant DNA, but not due to thechromosomal location of the integration of the transgene. Twelveindependent transgenic lines for each recombinant DNA construct wereestablished and assayed for the transgene expression levels. Fivetransgenic lines with high transgene expression levels were used in thefirst pass screen to evaluate the transgene's function in T2 transgenicplants. Subsequently, three transgenic events, which had been shown tohave one or more improved traits, were further evaluated in the secondpass screen to confirm the transgene's ability to impart an improvedtrait. The following Table 2 summarizes the improved traits that havebeen confirmed as provided by a recombinant DNA construct.

In particular, Table 2 reports

“PEP SEQ ID” which is the amino acid sequence of the protein cognate tothe DNA in the recombinant DNA construct corresponding to a proteinsequence of a SEQ ID NO. in the Sequence Listing.

“construct_id” is an arbitrary name for the recombinant DNA describemore particularly in Table 1.

“annotation” refers to a description of the top hit protein obtainedfrom an amino acid sequence query of each PEP SEQ ID NO to GenBankdatabase of the National Center for Biotechnology Information (ncbi).More particularly, “gi” is the GenBank ID number for the top BLAST hit.

“description” refers to the description of the top BLAST hit.

“e-value” provides the expectation value for the BLAST hit.

“identity” refers to the percentage of identically matched amino acidresidues along the length of the portion of the sequences which isaligned by BLAST between the sequence of interest provided herein andthe hit sequence in GenBank.

“traits” identified by two letters codes the confirmed improvement in atransgenic plant provided by the recombinant DNA. The codes for improvedtraits are:

“CK” which indicates cold tolerance improvement identified under a coldshock tolerance screen;

“CS” which indicates cold tolerance improvement identified by a coldgermination tolerance screen;

“DS” which indicates drought tolerance improvement identified by a soildrought stress tolerance screen;

“PEG” which indicates osmotic stress tolerance improvement identified bya PEG induced osmotic stress tolerance screen;

“HS” which indicates heat stress tolerance improvement identified by aheat stress tolerance screen;

“SS” which indicates high salinity stress tolerance improvementidentified by a salt stress tolerance screen;

“LN” which indicates nitrogen use efficiency improvement identified by alimited nitrogen tolerance screen;

“LL” which indicates attenuated shade avoidance response identified by ashade tolerance screen under a low light condition;

“PP” which indicates improved growth and development at early stagesidentified by an early plant growth and development screen;

“SP” which indicates improved growth and development at late stagesidentified by a late plant growth and development screen providedherein.

TABLE 2 Pep annotation SEQ construct e % Id id gene value identity ncbiid description traits 270 14324 CGPG1560 1.00E−127 86 gi|15232185|ref|NP_191546.1|expressed CK SS protein [Arabidopsis thaliana]] 27117484 CGPG2630 0 93 gi|15220912| ref|NP_173239.1|zinc finger CK(C3HC4-type RING finger) family protein [Arabidopsis thaliana] 272 19109CGPG1381 9.00E−31  81 gi|18404521| ref|NP_565870.1|expressed CK protein[Arabidopsis thaliana] 273 70423 CGPG3165 1.00E−134 96 gi|30688808|ref|NP_850953.1|MADS- CK CS CK HS PP box protein (AGL9) [Arabidopsisthaliana] gb|AAM65812.1| putative floral homeotic protein, AGL9[Arabidopsis thaliana] 274 70424 CGPG3180 1.00E−142 81 gi|25405039|pir||H96827protein CK F20B17.12 [imported] - Arabidopsis thalianagb|AAF68121.1| F20B17.12 [Arabidopsis thaliana] 275 70480 CGPG38331.00E−122 99 gi|18411867| ref|NP_565174.1|14-3-3 CK protein GF14 pi(GRF13) [Arabidopsis thaliana] 276 70509 CGPG2420 1.00E−113 82gi|15225186| ref|NP_180770.1|ovate CK protein-related [Arabidopsisthaliana] 277 70647 CGPG4334 1.00E−171 94 gi|15237269|ref|NP_200093.1|ornithine CK cyclodeaminase/mu- crystallin familyprotein [Arabidopsis thaliana] dbj|BAB10429.1| 278 70675 CGPG4519 0 100gi|15224730| ref|NP_180115.1|2- CK oxoglutarate-dependent dioxygenase,putative [Arabidopsis thaliana] pir||E84648 probable dioxygenase] 27970829 CGPG518 0 92 gi|15232841| ref|NP_186854.1|potassium CK transporter(KUP3) [Arabidopsis thaliana] 280 70849 CGPG596 1.00E−166 96gi|15224801| ref|NP_179547.1|cytidine CK deaminase (CDD)/cytidineaminohydrolase [Arabidopsis thaliana] 281 71627 CGPG1270 0 99gi|18398032| ref|NP_566315.1|ABC1 CK family protein [Arabidopsisthaliana] 282 71934 CGPG2294 1.00E−154 79 gi|15233973|ref|NP_195575.1|26S CK proteasome regulatory subunit S5A (RPN10)[Arabidopsis thaliana] sp|P55034|PSD4_ARATH 26S proteasome non- ATPaseregulatory subunit 4 (26S proteasome regulatory 283 72615 CGPG48292.00E−49  88 gi|18422886| ref|NP_568693.1|expressed CK protein[Arabidopsis thaliana] 284 72927 CGPG1477 1.00E−114 81 gi|15234815|ref|NP_194797.1|MA3 CK domain-containing protein [Arabidopsis thaliana]pir||A85359 translation initiation factor-like protein 285 73014CGPG5692 1.00E−180 93 gi|37528369| ref|NP_931714.1|Fructose- CK PP1,6-bisphosphatase (D- fructose-1,6-bisphosphate 1-phosphohydrolase)(FBPase) [Photorhabdus luminescens subsp. laumondii TTO1] 286 73559CGPG6535 0 93 gi|16078422| ref|NP_389241.1|similar to CK aspartateaminotransferase [Bacillus subtilis] sp|O31665|MTNE_BACSU TransaminasemtnE pir||F69863 probable transaminase (EC 2.6.1.—) ykrV 287 74251CGPG5489 1.00E−171 87 gi|15238437| ref|NP_200760.1|zinc CK SPtransporter (ZIP2) [Arabidopsis thaliana] sp|Q9LTH9|ZIP2_ARATH Zinctransporter 2 precursor (ZRT/IRT-like protein 2) 288 19631 CGPG36271.00E−94  90 gi|18410249| ref|NP_565053.1|SNF7 CS family protein[Arabidopsis thaliana] pir||G96755 developmental protein homolog DG1118[imported] - Arabidopsis thaliana 289 70121 CGPG2380 1.00E−111 100gi|6323765| ref|NP_013836.1|Hypothetical CS ORF; Ymr118cp [Saccharomycescerevisiae] sp|Q04487|YM07_YEAST Putative succinate dehydrogenasecytochrome B subunit, mitochondrial precursor 290 70654 CGPG43522.00E−63  88 gi|18400941| ref|NP_566531.1|expressed CS protein[Arabidopsis thaliana] 291 70696 CGPG4590 8.00E−92  87 gi|25408379|pir||E84768hypothetical CS PP protein At2g35430 292 70713 CGPG1462 0 95gi|30678679| ref|NP_191966.2|malate CS oxidoreductase, putative 29370740 CGPG3700 0 94 gi|18402759| ref|NP_566667.1|transcription CS LL PPfactor jumonji (jmjC) domain-containing protein 294 71321 CGPG4418 0 91gi|13878402| sp|Q9STL0|C71N_ARATH CS Cytochrome P450 71A23 pir||T06712probable cytochrome P450 T29H11.180 295 71835 CGPG4634 0 100gi|15234361| ref|NP_192100.1|DC1 CS SP domain-containing protein[Arabidopsis thaliana] pir||E85024 probable CHP- rich zinc fingerprotein 296 72934 CGPG5798 0 98 gi|6323512| ref|NP_013583.1|High- CSaffinity inorganic phosphate (Pi) transporter and low- affinitymanganese transporter; regulated by Pho4p and Spt7p; mutation confersresistance to arsenate; exit from the ER during maturation requiresPho86p; Pho84p [Saccharomyces cerevisiae] 297 72945 CGPG5787 0 94gi|6319991| ref|NP_010071.1|GABA- CS specific transport protein; Uga4p[Saccharomyces cerevisiae] sp|P32837|UGA4_YEAST GABA-specific permease(GABA-specific transport protein) 298 72980 CGPG5773 0 89 gi|6321960|ref|NP_012036.1|Subunit of CS the anaphase-promoting complex/cyclosome(APC/C), which is a ubiquitin-protein ligase required for degradation ofanaphase inhibitors, including mitotic cyclins, during themetaphase/anaphase transition; Cdc23p 299 73504 CGPG6480 1.00E−178 100gi|16330153| ref|NP_440881.1|fructokinase CS PP [Synechocystis sp. PCC6803] pir||S77227 fructokinase (EC 2.7.1.4) - Synechocystis sp (strainPCC 6803) 300 73507 CGPG6504 1.00E−178 100 gi|16078547|ref|NP_389366.1|similar to CS LL PP PEG glutaminase [Bacillus subtilis]301 73573 CGPG6462 0 100 gi|15890038| ref|NP_355719 1|AGR_C_5 CS PP 067p[Agrobacterium tumefaciens str. C58] ref|NP_533456.1| 3- isopropylmalatedehydrogenase 302 73586 CGPG6471 1.00E−177 100 gi|16077684|ref|NP_388498.1|similar to CS DS PP fructokinase [Bacillus subtilis] 30373770 CGPG5435 2.00E−34  68 gi|15235771| ref|NP_193383.1|cysteine CS PPprotease inhibitor family protein/cystatin family protein [Arabidopsisthaliana] 304 74105 CGPG6574 0 73 gi|23059330| ref|ZP_00084307.1|COG10CS 12: NAD-dependent aldehyde dehydrogenases [Pseudomonas fluorescensPfO-1] 305 74111 CGPG6622 0 99 gi|16131442| ref|NP_418028.1|alpha- CSamylase [Escherichia coli K12] 306 74136 CGPG6632 9.00E−81  99gi|16330993| ref|NP_441721.1|unknown CS CK HS protein [Synechocystis sp.PCC 6803] 307 74139 CGPG6561 1.00E−180 95 gi|24112825|ref|NP_707335.1|glyceraldehyde- CS LL 3-phosphate dehydrogenase A[Shigella flexneri 2a str. 301] 308 74267 CGPG5364 0 97 gi|18399375|ref|NP_566402.1|U-box CS domain-containing protein [Arabidopsisthaliana] 309 74291 CGPG5363 0 94 gi|18401867|ref|NP_565676.1|armadillo/ CS beta-catenin repeat family protein/U-boxdomain- containing protein [Arabidopsis thaliana] 310 74318 CGPG5826 0100 gi|15219730| ref|NP_176847.1|cell CS HS division protein kinase,putative [Arabidopsis thaliana] 311 74319 CGPG5831 0 96 gi|15224359|ref|NP_181907.1|mitogen- CS activated protein kinase, putative/MAPK,putative (MPK6) [Arabidopsis thaliana] 312 74324 CGPG5885 1.00E−174 95gi|42569304| ref|NP_180094.2|protein CS kinase family protein[Arabidopsis thaliana] 313 74512 CGPG32 0 96 gi|15217945|ref|NP_176132.1|amino CS HS SP acid permease T(AAP1) [Arabidopsisthaliana] 314 74583 CGPG6649 1.00E−151 83 gi|22978283|ref|ZP_00024043.1|COG02 CS PP 52: L- asparaginase/archaeal Glu- tRNAGlnamidotransferase subunit D [Ralstonia metallidurans] 315 70427 CGPG30670 100 gi|42572771| ref|NP_974481.1|kelch CS DS repeat-containing F-boxfamily protein [Arabidopsis thaliana] 316 71811 CGPG4426 0 97gi|15223341| ref|NP_171627.1|cytochrome CS DS LL LN P450, putative[Arabidopsis thaliana] 317 73463 CGPG6384 0 100 gi|22977164|ref|ZP_00022985.1|COG05 DS 38: Isocitrate dehydrogenases [Ralstoniametallidurans] 318 72081 CGPG5279 7.00E−66  77 gi|42570373|ref|NP_850277.2|CCAAT- DS PEG box binding transcription factor, putative[Arabidopsis thaliana] 319 10139 CGPG101 0 89 gi|15229877|ref|NP_187154.1|sodium DS proton exchanger, putative (NHX2) [Arabidopsisthaliana] 320 11410 CGPG103 0 82 gi|15236418| ref|NP_192555.1|homeoboxDS protein knotted-1 like 1 (KNAT1) [Arabidopsis thaliana] 321 11604CGPG48 0 92 gi|15233457| ref|NP_194642.1|hexokinase DS 1 (HXK1)[Arabidopsis thaliana] 322 12368 CGPG1006 1.00E−146 85 gi|15231451|ref|NP_190238.1|epsin N- DS terminal homology (ENTH) domain-containingprotein/ clathrin assembly protein- related [Arabidopsis thaliana] 32313502 CGPG1354 0 95 gi|15224557| ref|NP_180632.1|serine/threonine DS PPprotein kinase, putative [Arabidopsis thaliana] 324 13745 CGPG15761.00E−112 84 gi|15222987| ref|NP_177749.1|hypothetical DS protein[Arabidopsis thaliana] gb|AAF17642.1| T23E18.15 [Arabidopsis thaliana]325 13821 CGPG1569 1.00E−155 85 gi|18416499| ref|NP_567716.1|expressedDS protein [Arabidopsis thaliana] 326 14240 CGPG1697 0 94 gi|15241302|ref|NP_197527.1|expressed DS protein [Arabidopsis thaliana] 327 14718CGPG1082 0 86 gi|18407200| ref|NP_566090.1|expressed DS protein[Arabidopsis thaliana] 328 17022 CGPG1774 1.00E−159 100 gi|15237803|ref|NP_197755.1|nodulin DS MtN3 family protein [Arabidopsis thaliana]329 17924 CGPG2882 3.00E−92  100 gi|15233350| ref|NP_192875.1|zincfinger DS (C3HC4-type RING finger) family protein (RHA1b) [Arabidopsisthaliana] 330 18259 CGPG3368 2.00E−94  88 gi|30685085|ref|NP_849549.1|zinc finger DS PP protein (LSD1) [Arabidopsis thaliana]331 19171 CGPG2952 0 91 gi|6320063| ref|NP_010143.1|plasma DS membraneglucose sensor; Rgt2p [Saccharomyces cerevisiae] 332 19201 CGPG2332 0 95gi|15233948| ref|NP_194205.1|protein DS kinase (AFC2) [Arabidopsisthaliana] sp|P51567|AFC2_ARATH Protein kinase 333 19317 CGPG36621.00E−151 91 gi|21232858| ref|NP_638775.1|conserved DS hypotheticalprotein [Xanthomonas campestris pv. campestris str. ATCC 33913] 33470417 CGPG3427 0 81 gi|18396278| ref|NP_566180.1|integral DS PP SPmembrane family protein [Arabidopsis thaliana] 335 70467 CGPG3785 0 100gi|15241416| ref|NP_196953.1|no apical DS meristem (NAM) family protein[Arabidopsis thaliana] 336 70806 CGPG712 0 100 gi|15218225|ref|NP_173010.1|cyclin, DS putative [Arabidopsis thaliana] 337 70818CGPG479 1.00E−157 92 gi|30691978| ref|NP_568508.2|bZIP DS transcriptionfactor family protein [Arabidopsis thaliana] 338 70820 CGPG655 0 93gi|15224342| ref|NP_181899.1|acyl-[acyl- DS carrier-protein] desaturase/stearoyl-ACP desaturase (SSI2) [Arabidopsis thaliana] 339 70919 CGPG40291.00E−169 73 gi|6996560| emb|CAB75429.1|oligouridylate DS bindingprotein [Nicotiana plumbaginifolia] 340 71623 CGPG4696 1.00E−150 95gi|15236511| ref|NP_192588.1|mitogen- DS activated protein kinase,putative [Arabidopsis thaliana] pir||T01835 serine/threonine-specificprotein kinase ARA.KIN homolog T15F16.3- Arabidopsis thaliana 341 71662CGPG4679 1.00E−173 91 gi|5929964| gb|AAD56659.1|malate DS dehydrogenase[Glycine max] 342 71693 CGPG4652 6.00E−86  56 gi|21553460|gb|AAM62553.1|snap25a DS [Arabidopsis thaliana] 343 72384 CGPG4639 0 98gi|1169548| sp|P38604|ERG7_YEASTLa- DS nosterol synthase(Oxidosqualene-lanosterol cyclase) (2,3- epoxysqualene--lanosterolcyclase) (OSC) gb|AAA64377.1| 2,3- oxidosqualene-lanosterol cyclase 34472439 CGPG5075 8.00E−57  90 gi|22331337| ref|NP_683594.1|NPR1/ DSNIM1-interacting protein 2 (NIMIN-2) 345 72619 CGPG4835 6.00E−83  88gi|15237317| ref|NP_200108.1|expressed DS protein [Arabidopsis thaliana]346 72624 CGPG4842 0 100 gi|15242148| ref|NP_200558.1|expressed DSprotein [Arabidopsis thaliana] 347 72715 CGPG5521 0 91 gi|6323933|ref|NP_014004.1|Carboxy- DS SS terminal domain (CTD) phosphatase,essential for dephosphorylation of the repeated C-terminal domain of theRNA polymerase II large subunit (Rpo21p); Fcp1p [Saccharomycescerevisiae] 348 72754 CGPG5548 1.00E−169 100 gi|728961|sp|Q00618|BET4_YEASTGe- DS ranylgeranyl transferase type II alphasubunit (Type II protein geranyl- 349 72819 CGPG4989 0 100 gi|18417026|ref|NP_567780.1|pfkB-type DS carbohydrate kinase family protein[Arabidopsis thaliana] 350 75516 CGPG7689 1.00E−138 70 gi|42568081|ref|NP_197938.2|zinc finger DS (C3HC4-type RING finger) family protein[Arabidopsis thaliana] 351 75701 CGPG7856 8.00E−41  37 gi|15225413|ref|NP_182037.1|zinc finger DS LN (C2H2 type) family protein[Arabidopsis thaliana] 352 73515 CGPG6473 1.00E−162 100 gi|16079626|ref|NP_390450.1|similar to HS CS PEG 6-phosphogluconate dehydrogenase(pentose phosphate) [Bacillus subtilis] 353 74684 CGPG6360 1.00E−64  100gi|18390735| ref|NP_563782.1|expressed CS HS protein [Arabidopsisthaliana] 354 19542 CGPG3069 0 90 gi|18403574| ref|NP_564592.1|F-box HSfamily protein [Arabidopsis thaliana] 355 19618 CGPG3574 1.00E−121 100gi|15218423| ref|NP_177373.1|trypsin HS and protease inhibitor familyprotein/Kunitz family protein [Arabidopsis thaliana] pir||F96746probable drought induced protein 356 19649 CGPG3140 1.00E−141 87gi|18412787| ref|NP_567287.1|vesicle- HS associated membrane familyprotein/VAMP family protein 357 19745 CGPG3973 2.00E−60  46 gi|15239303|ref|NP_201424.1|expressed HS protein (Arabidopsis thaliana] 358 19768CGPG4096 1.00E−179 81 gi|25052804| gb|AAN65180.1|mitogen- HS SSactivated protein kinase 4 [Petroselinum crispum] 359 19772 CGPG39393.00E−82  79 gi|7488744| pir||T09700MADS-box HS protein - alfalfa(fragment) gb|AAB51377.1| MADS-box protein [Medicago sativa] 360 19779CGPG4113 1.00E−153 89 gi|30681126| ref|NP_196201.2|phosphate CS HStranslocator-related [Arabidopsis thaliana] 361 19833 CGPG4074 1.00E−10779 gi|6683777| gb|AAF23363.1|CAGL2 CS HS PP [Cucumis sativus] 362 19862CGPG3961 2.00E−89  56 gi|15229637| ref|NP_188469.1|no apical HS meristem(NAM) family protein [Arabidopsis thaliana] dbj|BAB01106.1| unnamedprotein product [Arabidopsis thaliana] 363 19879 CGPG4009 0 75gi|18401703| ref|NP_564504.1|protein HS CS SS phosphatase 2C-related/PP2C-related [Arabidopsis thaliana] 364 70445 CGPG3728 2.00E−51  88gi|30696602| ref|NP_200357.2|protease HS inhibitor/seed storage/lipidtransfer protein (LTP) family protein [Arabidopsis thaliana] 365 70738CGPG3195 1.00E−96  100 gi|15234797| ref|NP_194791.1|expressed HS PPprotein [Arabidopsis thaliana] 366 71437 CGPG4043 1.00E−164 81gi|15241535| ref|NP_196433.1|serine/ HS threonine protein kinase,putative [Arabidopsis thaliana] 367 71572 CGPG4520 3.00E−83  92gi|18403850| ref|NP_565804.1|expressed HS protein [Arabidopsis thaliana]368 71617 CGPG1227 0 100 gi|15236219| ref|NP_195218.1|1- HS CKphosphatidylinositol phosphodiesterase-related [Arabidopsis thaliana]369 72532 CGPG4780 1.00E−118 90 gi|15236659| ref|NP_194120.1|expressedHS protein [Arabidopsis thaliana] 370 72757 CGPG5572 0 89 gi|15242402|ref|NP_197088.1|zinc finger HS LL PEG protein CONSTANS (CO) [Arabidopsisthaliana] 371 73412 CGPG6448 0 99 gi|28867589| ref|NP_790208.1|glutamineHS synthetase, type I [Pseudomonas syringae pv. tomato str. DC3000] 37274102 CGPG6550 1.00E−167 94 gi|15614187| ref|NP_242490.1|L- HSasparaginase [Bacillus halodurans C-125] 373 72633 CGPG4853 1.00E−145 86gi|15238013| ref|NP_199519.1|casein CS LL PEG kinase II beta chain,putative [Arabidopsis thaliana] 374 72456 CGPG4745 1.00E−75  92gi|15239846| ref|NP_196763.1|17.6 kDa DS LL class II heat shock protein(HSP17.6-CII) [Arabidopsis thaliana] 375 72963 CGPG1746 1.00E−151 87gi|15222239| ref|NP_172174.1|ovate LL LN family protein [Arabidopsisthaliana] 376 70426 CGPG3199 8.00E−54  88 gi|18397268|ref|NP_564336.1|double- LL stranded DNA-binding family protein[Arabidopsis thaliana] 377 70772 CGPG4627 1.00E−92  80 gi|15220084|ref|NP_173175.1|MADS- LL box protein (AGL100) [Arabidopsis thaliana] P378 71137 CGPG125 1.00E−111 90 gi|15218957| ref|NP_176202.1|two- LLcomponent responsive regulator/response regulator 3 (ARR3) [Arabidopsisthaliana] 379 71529 CGPG2808 1.00E−131 72 gi|42562375|ref|NP_174152.3|Dof-type LL zinc finger domain- containing protein[Arabidopsis thaliana] 380 71601 CGPG1858 1.00E−168 92 gi|15231425|ref|NP_187378.1|transcriptional LL activator, putative [Arabidopsisthaliana] 381 72362 CGPG983 1.00E−163 95 gi|15242779|ref|NP_200562.1|xylo- LL glucan:xyloglucosyl transferase,putative/xyloglucan endotransglycosylase, putative/endo-xyloglucantransferase, putative [Arabidopsis thaliana] 382 72466 CGPG47673.00E−60  83 gi|15234046| ref|NP_195030.1|glutaredoxin CK LL PEG familyprotein [Arabidopsis thaliana] 383 72524 CGPG4770 1.00E−134 91gi|18412649| ref|NP_567140.1|expressed CK LL protein [Arabidopsisthaliana] 384 73085 CGPG5689 1.00E−134 100 gi|16331347|ref|NP_442075.1|triosephos- LL phate isomerase [Synechocystis sp. PCC6803] 385 74241 CGPG5457 0 89 gi|444790| prf||1908224Anucleotide LLtranslocator 386 74247 CGPG5475 1.00E−159 100 gi|18411863|ref|NP_565172.1|protein LL phosphatase 2C, putative/ PP2C, putative[Arabidopsis thaliana] 387 74284 CGPG5413 0 97 gi|15230577|ref|NP_190087.1|serine LL carboxypeptidase III, putative [Arabidopsisthaliana] 388 74652 CGPG6168 1.00E−90  80 gi|15235970|ref|NP_194879.1|expressed LL DS protein [Arabidopsis thaliana] 389 70437CGPG3706 1.00E−172 100 gi|30678824| ref|NP_186983.2|short- CK LN chaindehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] 39071633 CGPG857 1.00E−100 86 gi|6690274| gb|AAF24061.1|v-SNARE DS LNAtVTI1a [Arabidopsis thaliana] 391 72948 CGPG5617 0 94 gi|15225456|ref|NP_182059.1|leucine- LN PEG rich repeat transmembrane proteinkinase, putative [Arabidopsis thaliana] 392 72519 CGPG4749 LN SS 39310475 CGPG399 1.00E−164 96 gi|15240972| ref|NP_195761.1|stress- LNresponsive protein, putative [Arabidopsis thaliana] 394 11120 CGPG459 0100 gi|15227169| ref|NP_179812.1|inositol-3- LN phosphate synthaseisozyme 2/myo-inositol-1- phosphate synthase 2/MI- 1-P synthase 2/IPS 2[Arabidopsis thaliana] 395 19736 CGPG4129 2.00E−94  67 gi|13346194|gb|AAK19619.1|GHMYB9 LN [Gossypium hirsutum] 396 71606 CGPG4715 0 91gi|15218674| ref|NP_171800.1|phototropic- LN responsive NPH3 familyprotein [Arabidopsis thaliana] 397 71840 CGPG4353 0 96 gi|18401087|ref|NP_566542.1|mitotic DS LL LN phosphoprotein N′ end (MPPN) familyprotein [Arabidopsis thaliana] 398 74240 CGPG5454 1.00E−155 90gi|15233884| ref|NP_194188.1|mitochondrial CK LN substrate carrierfamily protein [Arabidopsis thaliana] pir||T05577 uncoupling proteinhomolog F22K18.230 - Arabidopsis thaliana 399 74331 CGPG5834 0 94gi|15220416| ref|NP_172003.1|protein LN kinase family protein[Arabidopsis thaliana] 400 74610 CGPG6048 1.00E−117 100 gi|15217568|ref|NP_172434.1|Ras- LL LN related GTP-binding protein, putative[Arabidopsis thaliana] sp|O04486|RB1C_ARATH Ras-related protein Rab11C401 75527 CGPG7682 1.00E−55  65 gi|15240946|ref|NP_195750.1|phosphatidyl- LN ethanolamine-binding family protein[Arabidopsis thaliana] 402 70681 CGPG4584 9.00E−64  93 gi|18411465|ref|NP_567196.1|auxin- CK PEG responsive family protein [Arabidopsisthaliana] 403 71663 CGPG4638 0 93 gi|21230153| ref|NP_636070.1|conservedCK PEG hypothetical protein [Xanthomonas campestris pv. campestris str.ATCC 33913] 404 72769 CGPG5573 0 100 gi|15225499|ref|NP_182075.1|cytochrome CK PEG P450, putative [Arabidopsis thaliana]405 71508 CGPG1541 2.00E−24  100 gi|15241504| sp|Q9SD80|OM05_ARATH PEGCS SP PEG Mitochondrial import receptor subunit TOM5 homolog(Translocase of outer membrane 5 kDa subunit homolog) 406 74248 CGPG54760 100 gi|15226152| ref|NP_180926.1|protein PEG CS phosphatase 2C,putative/ PP2C, putative [Arabidopsis thaliana] p 407 72771 CGPG21665.00E−44  100 gi|18395032| ref|NP_564151.1|expressed PEG CK HS SSprotein [Arabidopsis thaliana] 408 72085 CGPG5228 0 94 gi|15241541|ref|NP_199275.1|cytochrome PEG HS P450 family protein [Arabidopsisthaliana] dbj|BAA98115.1|flavonoid 3′,5′-hydroxylase-like; cytochromeP450 [Arabidopsis thaliana] 409 72744 CGPG5563 1.00E−136 96 gi|6321574|ref|NP_011651.1|20S HS PEG CK proteasome beta-type subunit; the onlynonessential 20S subunit; Pre9p [Saccharomyces cerevisiae] 410 73039CGPG810 0 96 gi|15242124| ref|NP_197599.1|molybdopterin HS PEGbiosynthesis CNX1 protein/molybdenum cofactor biosynthesis enzyme CNX1(CNX1) [Arabidopsis thaliana] 411 73054 CGPG5754 2.00E−98  100gi|6324827| ref|NP_014896.1|Nat5p PEG HS SS PEG [Saccharomycescerevisiae] pir||S67150 hypothetical protein YOR253w - yeast(Saccharomyces cerevisiae) 412 73501 CGPG6456 0 97 gi|15888752|ref|NP_354433.1|AGR_C_2 HS PEG 631p [Agrobacterium tumefaciens str. C58]sp|Q8UFH1|ENO_AGRT5 Enolase (2- phosphoglycerate dehydratase)(2-phospho- D-glycerate hydro-lyase) 413 19707 CGPG4179 1.00E−86  49gi|15236282| ref|NP_195242.1|O- PEG CS methyltransferase family 2protein [Arabidopsis thaliana] 414 19951 CGPG3941 5.00E−91  54gi|15221582| ref|NP_177064.1|basic PEG CK helix-loop-helix (bHLH) familyprotein [Arabidopsis thaliana] 415 19967 CGPG4032 1.00E−127 67gi|4760710| dbj|BAA77395.1|SLL2-S9- PEG protein [Brassica rapa] 41670543 CGPG3815 0 95 gi|15220994| ref|NP_175222.1|E2F PEG CKtranscription factor-2 (E2F2)/transcription factor E2Fc (E2Fc)[Arabidopsis thaliana] 417 70707 CGPG1273 1.00E−108 100 gi|15219558|ref|NP_177523.1|Ssu72- PEG like family protein [Arabidopsis thaliana]pir||F96765 unknown protein F 418 70719 CGPG1712 0 86 gi|18394560|ref|NP_564043.1|expressed PEG protein [Arabidopsis thaliana] 419 71134CGPG817 8.00E−55  100 gi|15240471| ref|NP_200327.1|small PEG HS PPubiquitin-like modifier 2 (SUMO) [Arabidopsis thaliana] 420 71146CGPG2928 1.00E−86  92 gi|29165403| gb|AAO65311.1|MADS PEG affectingflowering 3 variant II [Arabidopsis thaliana] 421 71660 CGPG46903.00E−80  100 gi|18415773| ref|NP_567637.1|methionine PEG sulfoxidereductase domain-containing protein/ SelR domain-containing protein[Arabidopsis thaliana] 422 72086 CGPG5236 0 100 gi|15232215|ref|NP_191556.1|methylene- PEG PP PEG tetrahydrofolate reductase 1(MTHFR1) [Arabidopsis thaliana] - 423 72632 CGPG4852 4.00E−99  90gi|18425032| ref|NP_569028.1|expressed PEG protein [Arabidopsisthaliana] 424 72716 CGPG5529 2.00E−85  89 gi|6320196|ref|NP_010276.1|subunit of PEG the Anaphase Promoting Complex; all knownAPC subunits co- immunoprecipitate with epitope-tagged Apc11p; Apc11p[Saccharomyces cerevisiae] 425 72723 CGPG1848 0 97 gi|15237500|ref|NP_199487.1|human PEG Rev interacting-like family protein/hRIPfamily protein [Arabidopsis thaliana] dbj|BAB08919.1| zinc fingerprotein Glo3-like [Arabidopsis thaliana] 426 72987 CGPG1787 5.00E−81  77gi|15231568| ref|NP_189282.1|octicosa- PEG SP peptide/Phox/Bem1p (PB1)domain-containing protein [Arabidopsis thaliana] 427 74109 CGPG6606 0 77gi|37524479| ref|NP_927823.1|maltodextrin PEG phosphorylase[Photorhabdus luminescens subsp. laumondii TTO1] 428 74140 CGPG6569 0 99gi|15613102| ref|NP_241405.1|NADP- PEG PP PEG dependent aldehydedehydrogenase [Bacillus halodurans C-125] 429 74191 CGPG6597 0 96gi|22960294| ref|ZP_00007935.1|COG18 PEG 50: Ribulose 1,5- bisphosphatecarboxylase, large subunit [Rhodobacter sphaeroides] 430 74265 CGPG53561.00E−117 100 gi|15237288| ref|NP_197727.1|GRAM PEG PP PEGdomain-containing protein/ ABA-responsive protein- related [Arabidopsisthaliana] 431 74369 CGPG6076 2.00E−86  96 gi|18409647|ref|NP_564994.1|ubiquitin- PEG CK PP PEG conjugating enzyme familyprotein [Arabidopsis thaliana] 432 70217 CGPG6 0 97 gi|15231536|ref|NP_189259.1|cytochrome CK PP SP P450 family protein [Arabidopsisthaliana] 433 72711 CGPG1846 4.00E−75  79 gi|15221048|ref|NP_175816.1|transcription CK PP SP initiation factor IID (TFIID) 31kDa subunit (TAFII-31) family protein [Arabidopsis thaliana] 434 70932CGPG4089 1.00E−129 56 gi|15223134| ref|NP_177792.1|expressed CS HS PPprotein [Arabidopsis thaliana] 435 73518 CGPG6497 1.00E−177 63gi|22981996| ref|ZP_00027327.1|COG10 CS CK PP 12: NAD-dependent aldehydedehydrogenases [Burkholderia fungorum] 436 19771 CGPG4011 3.00E−90  80gi|18418200| ref|NP_568342.1|rubredoxin PP HS SS family protein[Arabidopsis thaliana] dbj|BAB10504.1| gene_id: MKP11.2~unknown protein[Arabidopsis thaliana] g 437 73549 CGPG6460 0 90 gi|37524978|ref|NP_928322.1|5- HS DS PP carboxymethyl-2- hydroxymuconatesemialdehyde dehydrogenase [Photorhabdus luminescens subsp. laumondiiTTO1] 438 72994 CGPG5803 0 83 gi|6322702| ref|NP_012776.1|Vacuolar CKPEG CS PP PEG transporter, exports large neutral amino acids from thevacuole; member of a family of seven S. cerevisiae genes (AVT1-7)related to vesicular GABA- glycine transporters; Avt3p [Saccharomycescerevisiae] 439 71928 CGPG1617 0 100 gi|18394888|ref|NP_564120.1|catalase 3 CS PEG CK PP PEG (SEN2) [Arabidopsisthaliana] 440 72903 CGPG5584 0 91 gi|6322293| ref|NP_012367.1|HistonePEG PP SS methyltransferase with a role in transcriptional elongation,methylates a lysine residue of histone H3; associates with the C-terminal domain of Rpo21p; histone methylation activity is regulated byphosphorylation status of Rpo21p; Set2p [Saccharomyces cerevisiae]sp|P46995|SET2_YEAST SET domain protein 2 441 73017 CGPG5733 0 94gi|6325368| ref|NP_015436.1|kinase PEG PP PEG required for late nucleardivision; Dbf20p [Saccharomyces cerevisiae] 442 74587 CGPG6774 0 94gi|17938451| ref|NP_535240.1|succinate PEG DS HS PP SS semialdehydedehydrogenase [Agrobacterium tumefaciens str. C58] 443 72453 CGPG47356.00E−67  91 gi|15218924| ref|NP_174236.1|auxin- CK PP SP SS responsivefamily protein [Arabidopsis thaliana] pir||A86417 probable auxin-induced protein, 45653- 45228 444 72967 CGPG5742 0 99 gi|6321525|ref|NP_011602.1|Cytosolic CS CK HS LL PP SS catalase T, has a role inprotection from oxidative damage by hydrogen peroxide; Ctt1p[Saccharomyces cerevisiae] 445 72961 CGPG5591 0 95 gi|15228498|ref|NP_86975.1|UTP-- PEG SS HS PP glucose-1-phosphateuridylyltransferase, putative/ UDP-glucose pyrophosphorylase,putative/UGPase, putative [Arabidopsis thaliana] 446 73070 CGPG5627 0 90gi|15225044| ref|NP_181451.1|protein PEG PP SS kinase family protein[Arabidopsis thaliana] 447 73475 CGPG6385 0 100 gi|39934021|ref|NP_946297.1|glyceraldehyde- PEG PP SS 3-phosphatedehydrogenase(GAPDH) [Rhodopseudomonas palustris CGA009] 448 72916CGPG1814 0 97 gi|15228871| ref|NP_188303.1|protein PP SS phosphatase 2C,putative/ PP2C, putative [Arabidopsis thaliana] 449 72969 CGPG5789 0 94gi|6321886| ref|NP_011962.1|Low- PP SP SS affinity glucose transporterof the major facilitator superfamily, expression is induced by Hxk2p inthe presence of glucose and repressed by Rgt1p when glucose is limiting;Hxt1p [Saccharomyces cerevisiae] 450 74449 CGPG6659 0 96 gi|15890426|ref|NP_356098.1|AGR_L_6 PP SS 19p [Agrobacterium tumefaciens str. C58]pir||A98170 hypothetical protein AGR_L_619 [imported] - Agrobacterium451 16615 CGPG2539 0 98 gi|15890896| ref|NP_356568.1|AGR_L_1 PP 560p[Agrobacterium tumefaciens str. C58] ref|NP_534561.1| glucose-1-phosphate adenylyltransferase [Agrobacterium tumefaciens str. C58] 45219187 CGPG3310 0 91 gi|18423163| ref|NP_568731.1|squamosa PPpromoter-binding protein, putative [Arabidopsis thaliana] 453 19648CGPG3134 1.00E−179 96 gi|18413950| ref|NP_568102.1|short- PP chaindehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] 45470864 CGPG3995 0 64 gi|15241312| ref|NP_196916.1|nodulin DS PP SP familyprotein [Arabidopsis thaliana] 455 70421 CGPG2946 0 88 gi|30677077|ref|NP_178317.2|zinc finger PP (C2H2 type) family protein [Arabidopsisthaliana] 456 70459 CGPG3758 0 95 gi|15233315| ref|NP_188242.1|F-box PPfamily protein [Arabidopsis thaliana] dbj|BAB01261.1| unnamed proteinproduct [Arabidopsis thaliana] 457 70465 CGPG3775 1.00E−155 90gi|15236937| ref|NP_195254.1|zinc finger PP SP (C2H2 type) familyprotein [Arabidopsis thaliana] 458 70683 CGPG4587 3.00E−65  64gi|18423239| ref|NP_568751.1|polyadenylate- PP binding protein,putative/PABP, putative [Arabidopsis thaliana] 459 70725 CGPG2097 0 91gi|18420505| ref|NP_568066.1|expressed CS PP protein [Arabidopsisthaliana] 460 70852 CGPG1465 0 93 gi|15237075|ref|NP_195290.1|isocitrate PP SP dehydrogenase, putative/ NAD+isocitrate dehydrogenase, putative [Arabidopsis thaliana] 461 71112CGPG934 1.00E−130 94 gi|15218701| ref|NP_171806.1|expressed CS PPprotein [Arabidopsis thaliana] pir||E86161 F10O3.11 protein -Arabidopsis thaliana gb|AAD25802.1| Belongs to the PF|01027Uncharacterized protein family UPF0005 with 7 transmembrane domains.[Arabidopsis thaliana] 462 71127 CGPG945 0 97 gi|15225307|ref|NP_179604.1|26S PP protease regulatory complex subunit 4, putative[Arabidopsis thaliana] pir||E84585 26S proteasome subunit 4 [imported] -Arabidopsis thaliana 463 71132 CGPG1561 0 98 gi|15232209|ref|NP_191550.1|expressed PP protein [Arabidopsis thaliana] 464 71217CGPG95 0 100 gi|15221476| ref|NP_172127.1|shaggy- PP related proteinkinase iota/ ASK-iota (ASK9) (GSK1) [Arabidopsis thaliana] (EC 2.7.1.—)465 71645 CGPG4688 3.00E−69  100 gi|18401105| ref|NP_566544.1|phospho-PP transfer family protein [Arabidopsis thaliana] 466 71726 CGPG3894 093 gi|15217677| ref|NP_171725.1|no apical HS PP meristem (NAM) familyprotein [Arabidopsis thaliana] 467 72432 CGPG4562 1.00E−144 92gi|20152540| emb|CAD29662.1|putative PP SP auxin response factor 23[Arabidopsis thaliana] 468 72450 CGPG4732 1.00E−170 100 gi|15238890|ref|NP_197366.1|zinc finger LL PP (C3HC4-type RING finger) familyprotein [Arabidopsis thaliana] 469 72455 CGPG4742 1.00E−144 93gi|15242893| ref|NP_200597.1|anthranilate PP PEG synthase beta subunit,putative [Arabidopsis thaliana] 470 72727 CGPG5522 1.00E−118 100gi|6324107| ref|NP_014177.1|functionally PP related to TFIIB, affectsstart site selection in vivo; Ssu72p [Saccharomyces cerevisiae] 47172817 CGPG4987 0 96 gi|30679158| ref|NP_567238.2|AAA-type PP ATPasefamily protein [Arabidopsis 472 72992 CGPG5777 0 90 gi|6324981|ref|NP_015049.1|S- PP PEG adenosylMethionine Permease; Sam3p[Saccharomyces cerevisiae] 473 73007 CGPG5760 0 93 gi|6320865|ref|NP_010944.1|One of PP three possible beta- subunits of the Snf1kinase complex, allows nuclear localization of the Snf1 kinase complexin the presence of a nonfermentable carbon source; contains glycogen-binding domain; Gal83p [Saccharomyces cerevisiae] 474 73073 CGPG5688 095 gi|16331010| ref|NP_441738.1|fructose PP 1,6-bisphosphatase[Synechocystis sp. PCC 6803] 475 73506 CGPG6496 0 96 gi|23062569|ref|ZP_00087347.1|COG10 PP 12: NAD-dependent aldehyde dehydrogenases[Pseudomonas fluorescens PfO-1] 476 74107 CGPG6590 0 95 gi|15965198|ref|NP_385551.1|PYRUVATE PP SS DEHYDROGENASE ALPHA2 SUBUNIT PROTEIN[Sinorhizobium meliloti 1021] 477 74117 CGPG6575 0 81 gi|37528116|ref|NP_931461.1|Phenylacet- CS PP aldehyde dehydrogenase (PAD)[Photorhabdus luminescens subsp. laumondii TTO1] 478 74131 CGPG6592 0 96gi|16329404| ref|NP_440132.1|transaldolase PP SS [Synechocystis sp. PCC6803) B - 479 74344 CGPG5929 1.00E−111 100 gi|15236410|ref|NP_193147.1|COP9 HS PP signalosome subunit, putative/CSN subunit,putative (CSN8) [Arabidopsis thaliana] 480 14320 CGPG1229 0 100gi|18418018| ref|NP_567894.1|expressed SP protein [Arabidopsis thaliana]481 16756 CGPG2117 1.00E−142 85 gi|18391249| ref|NP_563885.1|expressedSP protein [Arabidopsis thaliana] 482 17448 CGPG2673 1.00E−102 72gi|15239624| ref|NP_197993.1|PHD SP finger family protein [Arabidopsisthaliana] gb|AAM64729.1| nucleic acid binding protein-like [Arabidopsisthaliana] 483 17633 CGPG2839 1.00E−145 85 gi|18395124|ref|NP_564171.1|basic SP helix-loop-helix (bHLH) family protein[Arabidopsis thaliana] 484 18876 CGPG3096 1.00E−172 89 gi|18394949|ref|NP_564133.1|transporter- SP related [Arabidopsis thaliana]pir||G86343 hypothetical protein T22I11.10 485 19120 CGPG1976 0 100gi|15232345| ref|NP_188710.1|fertilization- SP independent endospermprotein (FIE) [Arabidopsis thaliana] 486 19221 CGPG2958 1.00E−159 78gi|30690446| ref|NP_182182.2|Dof zinc SP finger protein DAG2/Dofaffecting germination 2 (DAG2) [Arabidopsis thaliana] 487 70206 CGPG41161.00E−139 64 gi|18412918| ref|NP_565249.1|phospholipid/ SP glycerolacyltransferase family protein [Arabidopsis 488 70223 CGPG53 0 93gi|15240313| ref|NP_198006.1|hexose SP transporter, putative[Arabidopsis thaliana] 489 70347 CGPG3147 1.00E−121 66 gi|18416267|ref|NP_567693.1|Dof-type SP zinc finger domain- containing protein[Arabidopsis thaliana] 490 70406 CGPG1687 0 93 gi|18397470|ref|NP_564354.1|early- SP responsive to dehydration stress protein(ERD4) [Arabidopsis thaliana] 491 70469 CGPG3791 1.00E−171 89gi|15237581| ref|NP_198936.1|MADS- SP HS box family protein [Arabidopsisthaliana]. 492 70564 CGPG1864 0 89 gi|15219067| ref|NP_173589.1|SWIRM SPdomain-containing protein/ DNA-binding family protein gb|AAD41423.1|Contains similarity to gb|AF033823 moira protein from Drosophilamelanogaster and contains a PF|00249 Myb-like DNA-binding domain. 49370601 CGPG2917 0 91 gi|15235140| ref|NP_193702.1|zinc finger SP PP(C3HC4-type RING finger) family protein [Arabidopsis thaliana]pir||T04748 hypothetical protein T16H5.30 - Arabidopsis thaliana 49470612 CGPG3721 0 96 gi|18416732| ref|NP_568256.1|conserved SP oligomericGolgi complex component-related/COG complex component-related[Arabidopsis thaliana] 495 70720 CGPG1358 0 93 gi|15238483|ref|NP_198387.1|lectin SP protein kinase family protein [Arabidopsisthaliana] 496 70735 CGPG2661 1.00E−109 100 gi|15231241|ref|NP_187953.1|transcription SP initiation factor IID-1(TFIID-1)/TATA-box factor 1/TATA sequence-binding protein 1 (TBP1)[Arabidopsis thaliana] 497 70846 CGPG377 1.00E−151 100 gi|15221223|ref|NP_177577.1|zinc finger SP (C3HC4-type RING finger) family protein[Arabidopsis thaliana] pir||D96772 probable RING zinc finger protein 49870923 CGPG4020 0 87 gi|8132347| gb|AAF73257.1|MAP SP kinase PsMAPK2[Pisum sativum] 499 71149 CGPG3457 0 83 gi|20141566|sp|P48001|HKL4_ARATHHo- SP meobox protein knotted-1 like 4 (KNAT4)pir||T51795 HOMEOBOX PROTEIN KNOTTED-1 LIKE 4 (KNAT4) - 500 71608CGPG4687 0 100 gi|15220438| ref|NP_172008.1|ent- SP kaurenoic acidhydroxylase (KAO1)/cytochrome P450 88A3, putative (CYP88A3) [Arabidopsisthaliana] 501 71739 CGPG4345 7.00E−89  80 gi|18406944|ref|NP_566061.1|expressed SP protein [Arabidopsis thaliana] 502 72014CGPG5230 0 100 gi|25410898| pir||D84423probable WD- SP 40-repeat protein[imported] - Arabidopsis thaliana gb|AAD14533.1| putative stress protein[Arabidopsis thaliana] 503 72051 CGPG5241 0 93 gi|18401606|ref|NP_566585.1|cyclic SP nucleotide-binding transporter 1/CNBT1(CNGC20) [Arabidopsis thaliana] sp|Q9LD37|CG20_ARATH Probable cyclicnucleotide- gated ion channel 20, chloroplast precursor (Cyclicnucleotide-binding transporter 1) 504 74259 CGPG5343 0 96 gi|15222882|ref|NP_175431.1|branched- CS HS SS chain amino acid aminotransferase 6/branched-chain amino acid transaminase 6 (BCAT6) [Arabidopsis thaliana]s 505 72463 CGPG4760 8.00E−48  100 gi|15236351| ref|NP_193115.1|auxin-CS SS HS LN PP responsive protein, putative [Arabidopsis thaliana] 50672902 CGPG5597 0 88 gi|15240576| ref|NP_199800.1|chloride SS CS DSchannel protein (CLC-c) [Arabidopsis thaliana] sp|Q96282|CLCC_ARATHChloride channel protein CLC-c (AtCLC-c) 507 74572 CGPG6640 1.00E−109 93gi|16331001| ref|NP_441729.1|unknown CS PP SS protein [Synechocystis sp.PCC 6803] 508 73055 CGPG5768 0 97 gi|6321588|ref|NP_011665.1|Hypothetical SS CS HS ORF; Ygr149wp [Saccharomycescerevisiae] 509 74103 CGPG6558 0 99 gi|15833050|ref|NP_311823.1|fructose- HS PP SS bisphosphate aldolase class II[Escherichia coli O157:H7] r 510 72921 CGPG5781 0 93 gi|6322892|ref|NP_012965.1|general CK PEG SS amino acid permease; Gap1p[Saccharomyces cerevisiae] 511 72968 CGPG5772 0 99 gi|6321546|ref|NP_011623.1|role in PEG LL SS DNA replication during S phase; Clb6p[Saccharomyces cerevisiae] 512 19703 CGPG4172 0 83 gi|7488676|pir||T07150G-box binding HS SS factor 2A - soybean (fragment)gb|AAB00097.1| G-box binding factor 513 19946 CGPG4097 1.00E−46  34gi|15219099| ref|NP_175691.1|2- SS oxoglutarate-dependent dioxygenase,putative (Arabidopsis thaliana] 514 19980 CGPG3914 2.00E−63  49gi|28629811| gb|AAO45179.1|transcription CS SS factor Myb1 [Malusxiaojinensis] 515 70435 CGPG3701 1.00E−150 91 gi|15236597|ref|NP_193499.1|casein SS PP SP kinase II beta chain, putative[Arabidopsis thaliana] 516 71114 CGPG1657 0 88 gi|30680729|ref|NP_849990.1|K + efflux SS antiporter, putative (KEA4) [Arabidopsisthaliana] 517 72451 CGPG4733 0 94 gi|15239622|ref|NP_197992.1|mitochondrial SS substrate carrier family protein[Arabidopsis thaliana] 518 72947 CGPG5607 3.00E−62  53 gi|1483230|emb|CAA67968.1|MADS4 SS protein [Betula pendula] 519 73012 CGPG5786 0 97gi|6324187| ref|NP_014257.1|belongs SS to a ubiquitous family ofcytoplasmic membrane proteins that transport only ammonium (NH(4)(+) +NH(3)).; Mep2p [Saccharomyces cerevisiae] 520 73022 CGPG5622 0 86gi|15225518| ref|NP_182083.1|protein SS kinase family protein[Arabidopsis thaliana] 521 73488 CGPG6394 1.00E−154 94 gi|16080620|ref|NP_391447.1|UTP- SS CS PP glucose-1-phosphate uridylyltransferase[Bacillus subtilis] 522 73901 CGPG5237 0 92 gi|18400284|ref|NP_565553.1|extra- SS large guanine nucleotide bindingprotein/G-protein (XLG) 523 73964 CGPG5804 0 88 gi|6319773|ref|NP_009855.1|Na+/Pi SS cotransporter, active in early growth phase;similar to phosphate transporters of Neurospora crassa; transcriptionregulated by inorganic phosphate concentrations and Pho4p; Pho89p [ 52474019 CGPG5706 2.00E−92  100 gi|16079815| ref|NP_390639.1|adenine SSphosphoribosyltransferase [Bacillus subtilis] 525 74022 CGPG5724 0 97gi|18378991| ref|NP_563659.1|glycosyl- SS SP hydrolase family 3 protein[Arabidopsis thaliana] 526 74114 CGPG6551 0 99 gi|15888903|ref|NP_354584.1|AGR_C_2 SS 921p [Agrobacterium tumefaciens str. C58]pir||H97551 probable aminotransferase aatc 527 74262 CGPG5353 0 100gi|18416245| ref|NP_568226.1|histidinol- SS PP phosphateaminotransferase, putative [Arabidopsis thaliana] 528 74292 CGPG5367 096 gi|15239204| ref|NP_201393.1|U-box SS domain-containing protein[Arabidopsis thaliana] 529 74302 CGPG5384 1.00E−59  82 gi|25313155|pir||A96787protein F10A5.6 PP SS [imported] - Arabidopsis thaliana 53074325 CGPG5898 1.00E−174 86 gi|15230382| ref|NP_188576.1|cinnamyl- SSalcohol dehydrogenase (CAD) [Arabidopsis thaliana] 531 74429 CGPG6689 096 gi|16077873| ref|NP_388687.1|acetoin SS dehydrogenase E1 component(TPP- dependent alpha subunit) [Bacillus subtilis] 532 74440 CGPG66826.00E−90  82 gi|15613838| ref|NP_242141.1|uridine SS kinase [Bacillushalodurans C-125] 533 74462 CGPG6668 5.00E−67  99 gi|16332127|ref|NP_442855.1|unknown HS SS protein [Synechocystis sp. PCC 6803] 53474465 CGPG6692 1.00E−119 99 gi|16078642| ref|NP_389461.1|similar to PPSS ribulose-5-phosphate 3- epimerase [Bacillus subtilis] 535 74474CGPG6669 2.00E−85  82 gi|16331209| ref|NP_441937.1|unknown LL SS protein[Synechocystis sp. PCC 6803] 536 74505 CGPG6783 0 100 gi|16129426|ref|NP_415984.1|cryptic SS nitrate reductase 2 beta subunit [Escherichiacoli K12] 537 74507 CGPG6799 0 82 gi|27479656| gb|AAO17183.1|Orf17 SP SS[Photorhabdus luminescens] 538 74562 CGPG6764 0 95 gi|16077501|ref|NP_388315.1|similar to SS pyruvate oxidase [Bacillus subtilis]

Screens for Identifying Trait Improving Genes

DS-Improvement of Drought Tolerance Identified by a Soil Drought StressTolerance Screen:

Drought is a water deficit condition that imposes osmotic stress onplants. Plants are particularly vulnerable to drought during theflowering stage. The drought condition in the screening processdisclosed in Example 1B started from the flowering time and wassustained to the end of harvesting. The drought tolerance-imparting DNAdefined for this invention are used in recombinant DNA constructs thatimprove plant survival rate under drought conditions. Exemplaryrecombinant DNA which has been identified for conferring such droughttolerance is identified as such in Table 2. Such identified recombinantDNA is useful in generating transgenic plants that are tolerant to thedrought condition imposed during flowering time and in other stages ofthe plant life cycle. As demonstrated from the model plant screen, insome embodiments of transgenic plants with trait-improving recombinantDNA grown under such sustained drought condition also have increasedtotal seed weight per plant in addition to the increased survival ratewithin a transgenic population, providing a higher yield potential ascompared to control plants.

PEG-Improvement of Drought Tolerance Identified by PEG Induced OsmoticStress Tolerance Screen:

Various drought levels can be artificially induced by using variousconcentrations of polyethylene glycol (PEG) to produce different osmoticpotentials (Pilon-Smits et al., (1995) Plant Physiol. 107:125-130).Several physiological characteristics have been reported as beingreliable indications for selection of plants possessing droughttolerance. These characteristics include the rate of seed germinationand seedling growth. The traits can be assayed relatively easily bymeasuring the growth rate of seedling in PEG solution. Thus, aPEG-induced osmotic stress tolerance screen is a useful surrogate fordrought tolerance screen. Certain embodiments of transgenic plants withtrait-improving recombinant DNA identified in the PEG-induced osmoticstress tolerance screen survive drought conditions providing a higheryield potential as compared to control plants.

SS-Improvement of Drought Tolerance Identified by High Salinity StressTolerance Screen:

Three different factors are responsible for salt damages: (1) osmoticeffects, (2) disturbances in the mineralization process, (3) toxiceffects caused by the salt ions, e.g., inactivation of enzymes. Whilethe first factor of salt stress results in the wilting of the plantsthat is similar to drought effect, the ionic aspect of salt stress isclearly distinct from drought. Exemplary recombinant DNA which has beenidentified to help plants maintain biomass, root growth and/or plantdevelopment in high salinity conditions are identified as such in Table2. Since osmotic effect is one of the major components of salt stress,which is common to the drought stress, embodiments of trait-improvingrecombinant DNA identified in a high salinity stress tolerance screenalso provide transgenic crops with improved drought tolerance.Embodiments of transgenic plants with trait-improving recombinant DNAidentified in a high salinity stress tolerance screen survive droughtconditions and/or high salinity conditions providing a higher yieldpotential as compared to control plants.

HS-Improvement of Drought Tolerance Identified by Heat Stress ToleranceScreen:

Heat and drought stress often occur simultaneously, limiting plantgrowth. Heat stress can cause the reduction in photosynthesis rate,inhibition of leaf growth and osmotic potential in plants. Thus, genesidentified as heat stress tolerance conferring genes may also impartimproved drought tolerance to plants. As demonstrated from the modelplant screen, embodiments of transgenic plants with trait-improvingrecombinant DNA identified in a heat stress tolerance screen can survivebetter heat stress conditions and/or drought conditions providing ahigher yield potential as compared to control plants.

CK and CS-Improvement of Tolerance to Cold Stress:

Low temperature may immediately result in mechanical constraints,changes in activities of macromolecules, and reduced osmotic potential.Two screening conditions, i.e., cold shock tolerance screen (CK) andcold germination tolerance screen (CS), were set up to look fortransgenic plants that display visual growth advantage at lowertemperature. In cold germination tolerance screen, the transgenicArabidopsis plants were exposed to a constant temperature of 8 degreesC. from planting until day 28 post planting. The trait-improvingrecombinant DNA identified by such screen are particular useful for theproduction of transgenic plant that can germinate more robustly in acold temperature as compared to the wild type plants. In cold shocktolerance screen, the transgenic plants were first grown under thenormal growth temperature of 22 degrees C. until day 8 post planting,and subsequently were placed under 8 degrees C. until day 28 postplanting. Embodiments of transgenic plants with trait-improvingrecombinant DNA identified in a cold shock stress tolerance screenand/or a cold germination stress tolerance screen survive coldconditions providing a higher yield potential as compared to controlplants.

Improvement of Tolerance to Multiple Stresses:

Different kinds of stresses often lead to identical or similar reactionin the plants. Genes that are activated or inactivated as a reaction tostress can either act directly in a way the genetic product reduces aspecific stress, or they can act indirectly by activating other specificstress genes. By manipulating the activity of such regulatory genes,i.e., multiple stress tolerance genes, plants are enabled to react todifferent kinds of stresses. For examples, DNA for expressing proteinsof SEQ ID NO:352 and SEQ ID NO:353 is useful to improve both heat stresstolerance and cold stress tolerance in plants. Plants transformed withDNa for expressing protein of SEQ ID NO:508 resist heat stress, saltstress and cold stress. Thus, the disclosed stress tolerance conferringgenes are useful in combinations to generate transgenic plants thatresist multiple stress conditions.

PP-Improvement of Early Plant Growth and Development:

It is known in the art that to minimize the impact of disease on cropprofitability, it is important to start the season with healthy vigorousplants. This means avoiding seed and seedling diseases, leading toincreased nutrient uptake and increased yield potential. Traditionallyearly planting and applying fertilizer are the methods used forpromoting early seedling vigor. In early development stage, plantembryos establish only the basic root-shoot axis, a cotyledon storageorgan(s), and stem cell populations, called the root and shoot apicalmeristems, that continuously generate new organs throughoutpost-embryonic development. “Early growth and development” encompassesthe stages of seed imbibition through the early vegetative phase.Certain DNA is identified as useful to produce transgenic plants thathave advantages in one or more processes including, but not limited to,germination, seedling vigor, root growth and root morphology undernon-stressed conditions. The transgenic plants starting from a morerobust seedling are less susceptible to the fungal and bacterialpathogens that attach germinating seeds and seedling. Furthermore,seedlings with advantage in root growth are more resistant to droughtstress due to extensive and deeper root architecture. Therefore, genesconferring the growth advantage in early stages to plants are used togenerate transgenic plants that are more resistant to various stressconditions due to improved early plant development. Exemplaryrecombinant DNA that confers both stress tolerance and growth advantagesto plants, is identified as such in Table 2, e.g., DNA encoding aprotein of SEQ ID NO:444 can improve the plant early growth anddevelopment, and impart heat and cold tolerance to plants. Embodimentsof transgenic plants with trait-improving recombinant DNA identified inthe early plant development screen grow better under non-stressconditions and/or stress conditions providing a higher yield potentialas compared to control plants.

SP-Improvement of Late Plant Growth and Development:

“Late growth and development” encompasses the stages of leafdevelopment, flower production, and seed maturity. Transgenic plantswith late growth and development advantages express DNA that isidentified as such in Table 2. Such plants exhibit at least onephenotypic characteristics including, but not limited to, increasedrosette radius, increased rosette dry weight, seed dry weight, siliquedry weight, and silique length. For example, the rosette radius androsette dry weight are used as the indexes of photosynthesis capacity,and thereby plant source strength and yield potential of a plant. Seeddry weight, silique dry weight and silique length are used as theindexes for plant sink strength, which are considered as the directdeterminants of yield. Embodiments of transgenic plants withtrait-improving recombinant DNA identified in the late developmentscreen grow better and/or have improved development during leafdevelopment and seed maturation providing a higher yield potential ascompared to control plants.

LL-Improvement of Tolerance to Shade Stress Identified in a Low LightScreen:

The effects of light on plant development are especially prominent atthe seedling stage. Under normal light conditions with unobstructeddirect light, a plant seeding develops according to a characteristicphotomorphogenic pattern, in which plants have open and expandedcotyledons and short hypocotyls. Then the plant's energy is devoted tocotyledon and leaf development while longitudinal extension growth isminimized. Under low light condition where light quality and intensityare reduced by shading, obstruction or high population density, aseedling displays a shade-avoidance pattern, in which the seedlingdisplays a reduced cotyledon expansion, and hypocotyls extension isgreatly increased. As the result, a plant under low light conditionincreases significantly its stem length at the expanse of leaf, seed orfruit and storage organ development, thereby adversely affecting ofyield. Recombinant DNA that enables plants to have an attenuated shadeavoidance response so that the source of plant can be contributed toreproductive growth efficiently provides embodiments of those plantswith higher yield as compared to the wild type plants. Embodiments oftransgenic plants with trait-improving recombinant DNA identified in ashade stress tolerance screen have attenuated shade response under shadeconditions providing a higher yield potential as compared to controlplants. The transgenic plants generated by this invention are suitablefor a higher density planting, thereby resulting increased yield perunit area.

LN-Improvement of Tolerance to Low Nitrogen Availability Stress

Nitrogen is a key factor in plant growth and crop yield. The metabolism,growth and development of plants are profoundly affected by theirnitrogen supply. Restricted nitrogen supply alters shoot to root ratio,root development, activity of enzymes of primary metabolism and the rateof senescence (death) of older leaves. All field crops have afundamental dependence on inorganic nitrogenous fertilizer. Sincefertilizer is rapidly depleted from most soil types, it must be suppliedto growing crops two or three times during the growing season. Enhancednitrogen use efficiency by plants should enable crops cultivated underlow nitrogen availability stress condition resulted from low fertilizerinput or poor soil quality.

Recombinant DNA that imparts enhanced nitrogen use efficiency intransgenic plants is identified in Table 2. Such plants exhibit one ormore desirable traits including, but not limited to, increased seedlingweight, increased number of green leaves, increased number of rosetteleaves, altered root length and advanced flower bud formation. Suchplants can also have altered amino acid or protein compositions,increased yield and/or better seed quality. Embodiments of suchtransgenic plants are productively cultivated under nitrogen nutrientdeficient conditions, i.e., nitrogen-poor soils and low nitrogenfertilizer inputs that cause the growth of wild type plants to cease orto be so diminished as to make the wild type plants practically uselessunder such conditions. The transgenic plants also are advantageouslyused to achieve earlier maturing, faster growing, and/or higher yieldingcrops and/or produce more nutritious foods and animal feedstocks whencultivated using nitrogen non-limiting growth conditions.

Stacked Traits:

This invention also provides transgenic plants with stacked engineeredtraits, e.g., a crop having an improved phenotype resulting fromexpression of a trait-improving recombinant DNA, in combination withherbicide and/or pest resistance traits. For example, genes of thecurrent invention can be stacked with other traits of agronomicinterest, such as a trait providing herbicide resistance, for example aglyphosate resistance trait, or insect resistance, such as using a genefrom Bacillus thuringiensis to provide resistance against lepidopteran,coliopteran, homopteran, hemiopteran, and other insects. Herbicides forwhich resistance is useful in a plant include glyphosate herbicides,phosphinothricin herbicides, oxynil herbicides, imidazolinoneherbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylureaherbicides, bialaphos herbicides, sulfonamide herbicides andgluphosinate herbicides. To illustrate that the production of transgenicplants with herbicide resistance is a capability of those of ordinaryskill in the art, reference is made to U.S. 2003-0106096 A1 and2002-0112260 A1 and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107,549 and6,376,754, all of which are incorporated herein by reference. Toillustrate that the production of transgenic plants with pest resistanceis a capability of those of ordinary skill in the art reference is madeto U.S. Pat. Nos. 5,250,515 and 5,880,275 which disclose plantsexpressing an endotoxin of Bacillus thuringiensis bacteria, to U.S. Pat.No. 6,506,599 which discloses control of invertebrates which feed ontransgenic plants which express dsRNA for suppressing a target gene inthe invertebrate, to U.S. Pat. No. 5,986,175 which discloses the controlof viral pests by transgenic plants which express viral replicase, andto U.S. Patent Application Publication 2003/0150017 A1 which disclosescontrol of pests by a transgenic plant which express a dsRNA targeted tosuppressing a gene in the pest, all of which are incorporated herein byreference.

Once one recombinant DNA has been identified as conferring an improvedtrait of interest in transgenic Arabidopsis plants, several methods areavailable for using the sequence of that recombinant trait-imparting DNAand knowledge about the protein it encodes to identify homologs of thatsequence from the same plant and different plant species or otherorganisms, e.g., bacteria and yeast. Thus, in one aspect, this inventionprovides methods for identifying a homologous gene with a DNA sequencehomologous to any of SEQ ID NO:1 through SEQ ID NO:269, or a homologousprotein with an amino acid sequence homologous to any of SEQ ID NO:270through SEQ ID NO:538. In another aspect, this invention provides aconsensus amino acid sequence for respective homologs for each of SEQ IDNO:270 through SEQ ID NO:538. In yet another aspect, this invention alsoincludes linking or associating one or more desired traits, or genefunction with a homolog sequence disclosed herein.

The trait-improving recombinant DNA and methods of using suchtrait-improving recombinant DNA for generating transgenic plants withimproved traits provided by this invention are not limited to anyparticular plant species. Indeed, the plants of this invention encompassmany species of monocots and dicots and include agriculturally usefulplants which are cultivated for purposes of food production orindustrial applications, e.g., corn and soybean plants and cottonplants. Recombinant DNA constructs optimized for soybean transformationand recombinant DNA constructs optimized for corn transformation aredisclosed in the following examples. Other plants of this inventioninclude canola, wheat, sunflower, sorghum, alfalfa, barley, millet,rice, tobacco, fruit and vegetable crops, and turfgrass.

Thus, embodiments of this invention include the use of both DNAidentified herein and homologs in recombinant DNA for transgenic cropplants with improved traits. Transgenic crop plants with improved traitsare identified from populations of plants grown from transgenic eventsby screening to segregate the plants of this invention from plantswithout the improved traits. Preferred screens for transgenic cropplants identify plants with improved responses to stress conditions,e.g., assays using imposed stress conditions to detect improvedresponses to drought stress, nitrogen deficiency, cold growingconditions, or alternatively, under naturally present stress conditions,for example under field conditions. Biomass measures are made ongreenhouse or field grown plants and include such measurements as plantheight, stem diameter, root and shoot dry weights, and, for corn plants,ear length and diameter.

Trait data on morphological changes is collected by visual observationduring the process of plant regeneration as well as in regeneratedplants transferred to soil. Such trait data includes characteristicssuch as normal plants, bushy plants, taller plants, thicker stalks,narrow leaves, striped leaves, knotted phenotype, chlorosis, albino,anthocyanin production, or altered tassels, ears or roots. Otherenhanced traits are identified by measurements taken under fieldconditions, such as days to pollen shed, days to silking, leaf extensionrate, chlorophyll content, leaf temperature, stand, seedling vigor,internode length, plant height, leaf number, leaf area, tillering, braceroots, stay green, stalk lodging, root lodging, plant health,barrenness/prolificacy, green snap, and pest resistance. In addition,trait characteristics of harvested grain are confirmed, including numberof kernels per row on the ear, number of rows of kernels on the ear,kernel abortion, kernel weight, kernel size, kernel density and physicalgrain quality.

To confirm hybrid yield in transgenic corn plants expressingtrait-imparting DNA of this invention, it is useful to test hybridplants over multiple years at multiple locations in a geographicallocation where corn is conventionally grown, e.g., in Iowa, Illinois andKansas, under “normal” field conditions as well as under stressconditions, e.g., under drought or population density stress.

Transgenic crop plants are used to provide other aspects of thisinvention such as transgenic seeds of crop plants. Seeds of transgenicplants are used to propagate more progeny plants which contain thetrait-improving recombinant DNA constructs of this invention. Theseprogeny plants are within the scope of this invention when they containa trait-improving recombinant DNA construct of this invention, whetheror not these plants are selfed or crossed with different varieties ofplants.

Screening Methods for Crop Transgenic Plants with Enhanced AgronomicTrait

Due to variability in transformation many transgenic events whichsurvive to fertile transgenic plants that produce seeds and progenyplants do not exhibit an enhanced agronomic trait. Thus, screening isnecessary to identify the transgenic events that produce the transgenicplants and seeds of this invention. Transgenic crop plants havingenhanced traits are identified from populations of plants transformed asdescribed herein by evaluating the trait in a variety of assays todetect an enhanced agronomic trait. Useful assays include analyses todetect changes in the chemical composition, biomass, physiologicalproperties and morphology of the plant. Changes in chemical compositionssuch as nutritional composition of grain are detected by analysis of theseed composition and content of protein, free amino acids, oil, freefatty acids, starch or tocopherols. Changes in biomass characteristicsare detected in greenhouse or field grown plants and include plantheight, stem diameter, root and shoot dry weights; and, for corn plants,ear length and diameter. Changes in physiological properties areidentified by evaluating responses to stress conditions, e.g., assaysusing imposed stress conditions such as water deficit, nitrogendeficiency, cold growing conditions, pathogen or insect attack or lightdeficiency, or increased plant density. Changes in morphology aremeasured by visual observation of tendency of a transformed plant withan enhanced agronomic trait to also appear to be a normal plant ascompared to changes toward bushy, taller, thicker, narrower leaves,striped leaves, knotted trait, chlorosis, albino, anthocyaninproduction, or altered tassels, ears or roots. Other screeningproperties include days to pollen shed, days to silking, leaf extensionrate, chlorophyll content, leaf temperature, stand, seedling vigor,internode length, plant height, leaf number, leaf area, tillering, braceroots, stay green, stalk lodging, root lodging, plant health,barrenness/prolificacy, green snap, and pest resistance. In addition,phenotypic characteristics of harvested grain are evaluated, includingnumber of kernels per row on the ear, number of rows of kernels on theear, kernel abortion, kernel weight, kernel size, kernel density andphysical grain quality.

Seeds for transgenic crop plants with enhanced agronomic traits of thisinvention are corn, soybean and cotton seeds, as well as seeds forcanola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice,tobacco, fruit and vegetable crops, and turfgrass.

A. Screening for Nitrogen Use Efficiency

Many transgenic crop plants of this invention exhibit enhanced nitrogenuse efficiency as compared to control plants. Higher nitrogen soilapplications increase seed protein and starch accumulation, and lead tolarger seed weight and larger kernel number per ear. Recent improvementsin elite high yielding corn hybrid genotypes include the ability toutilize nitrogen efficiently. DNA causing the enhanced nitrogen useefficiency in crop plants are especially useful, e.g., for improvingyield. Enhanced nitrogen use efficiency is assessed by measuring changesin plant growth such as leaf area production, shoot biomass, chlorophyllcontent in plants grown in nitrogen limiting conditions and/or nitrogensufficient conditions. It is useful to conduct a first screen innitrogen limiting conditions and confirm replicate transgenic events inboth nitrogen limiting and nitrogen sufficient conditions. Table 3 showsan amount of nutrients in the nutrient solution for nitrogen limitingconditions (low N) and nitrogen sufficient conditions (high N) which areuseful for nitrogen use efficiency screening. For example in agreenhouse screen pots of transgenic plants and control plants aretreated with 100 ml of nutrient solution three times a week on alternatedays starting at 8 and 10 days after planting for high N and low Nscreening, respectively.

TABLE 3 2 mM NH₄NO₃ 20 mM NH₄NO₃ Nutrient stock Low nitrogen Highnitrogen 1M NH₄N0₃ 2 mL/L 20 mL/L 1M KH₂PO₄ 0.5 0.5 1M MgSO₄•7H₂O 2 2 1MCaCl₂ 2.5 2.5 1M K₂SO₄ 1 1 Note: Adjust pH to 5.6 with HCl or KOH

After 28 days of plant growth for low N screening and 23 days for high Nscreening, measurements are taken for total shoot fresh mass, leafchlorophyll, leaf area, leaf fresh mass and leaf dry mass.

B. Screening for Increased Yield

Many transgenic plants of this invention exhibit improved yield ascompared to a control plant. Improved yield can result from a variety orother traits such as enhanced seed sink potential, e.g., the number andsize of endosperm cells or kernels, and/or enhanced sink strength, e.g.,the rate of starch biosynthesis. Sink potential is established veryearly during kernel development, as endosperm cell number and cell sizeare determined within the first few days after pollination.

Much of the increase in corn yield of the past several decades hasresulted from an increase in planting density. During that period, cornyield has been increasing at a rate of 2.1 bushels/acre/year, but theplanting density has increased at a rate of 250 plants/acre/year. Acharacteristic of modern hybrid corn is the ability of these varietiesto be planted at high density. Many studies have shown that a higherthan current planting density should result in more biomass production,but current germplasm does not perform well at these higher densities.One approach to increasing yield is to increase harvest index (HI), theproportion of biomass that is allocated to the kernel compared to totalbiomass, in high density plantings.

Effective yield screening of transgenic corn uses hybrid progeny of thetransgenic event over multiple locations with plants grown under optimalproduction management practices, and maximum pest control. A usefultarget for improved yield is a 5% to 10% increase in yield as comparedto yield produced by plants grown from seed for a control plant. Usefulscreening in multiple and diverse geographic locations, e.g., up to 16or more locations, over one or more planting seasons, e.g., at least twoplanting seasons, is useful to statistically distinguish yieldimprovement from natural environmental effects. Useful hybrid screeningincludes planting multiple transgenic plants, positive and negativecontrol plants, and pollinator plants in standard plots, e.g., 2 rowplots, 20 feet long by 5 feet wide with 30 inches distance between rowsand a 3 foot alley between ranges. Plants from separate transgenicevents can be grouped by recombinant DNA constructs with groups randomlyplaced in the field. A pollinator plot of a high quality corn line isplanted for every two plots to allow open pollination when using malesterile transgenic events. A useful planting density is about 30,000plants/acre.

Surrogate indicators for screening for yield improvement include sourcecapacity (biomass), source output (sucrose and photosynthesis), sinkcomponents (kernel size, ear size, starch in the seed), development(light response, height, density tolerance), maturity, early floweringtrait and physiological responses to high density planting, e.g., at45,000 plants per acre.

When screening for yield improvement a useful statistical measurementapproach comprises three components, i.e., modeling spatialautocorrelation of the test field separately for each location,adjusting traits of recombinant DNA events for spatial dependence foreach location, and conducting an across location analysis.

A first step in modeling spatial autocorrelation is estimating thecovariance parameters of the semivariogram. A spherical covariance modelis assumed to model the spatial autocorrelation. Because of the size andnature of the trial, it is likely that the spatial autocorrelation maychange. Therefore, anisotropy is also assumed along with sphericalcovariance structure. The following set of equations describes thestatistical form of the anisotropic spherical covariance model.

${{C\left( {h;\theta} \right)} = {{{vI}\left( {h = 0} \right)} + {{\sigma^{2}\left( {1 - {\frac{3}{2}h} + {\frac{1}{2}h^{3}}} \right)}{I\left( {h < l} \right)}}}},$where I(⋅) is the indicator function, h=√{square root over ({dot over(x)}²+{dot over (y)}²)}, and{dot over (x)}=[cos(ρπ/180)(x ₁-x ₂)−sin(ρπ/180)(y ₁-y ₂)]/ω_(x){dot over (y)}=[sin(ρπ/180)(x ₁-x ₂)+cos(ρπ/180)(y ₁-y ₂)]/ω_(y)where s₁=(x₁, y₁) are the spatial coordinates of one location ands₂=(x₂, y₂) are the spatial coordinates of the second location. Thereare 5 covariance parameters, θ=(ν, σ², ρ, ω_(n), ω_(j)), where ν is thenugget effect, σ² is the partial sill, ρ is a rotation in degreesclockwise from north, Ω_(n) is a scaling parameter for the minor axisand ω_(j) is a scaling parameter for the major axis of an anisotropicalellipse of equal covariance. The five covariance parameters that definethe spatial trend will then be estimated by using data from heavilyreplicated pollinator plots via restricted maximum likelihood approach.In a multi-location field trial, spatial trend are modeled separatelyfor each location.

After obtaining the variance parameters of the model, avariance-covariance structure is generated for the data set to beanalyzed. This variance-covariance structure contains spatialinformation required to adjust yield data for spatial dependence. Inthis case, a nested model that best represents the treatment andexperimental design of the study is used along with thevariance-covariance structure to adjust the yield data. During thisprocess the nursery or the seed batch effects can also be modeled andestimated to adjust the yields for any yield parity caused by seed batchdifferences.

After spatially adjusted data from different locations are generated,all adjusted data is combined and analyzed assuming locations asreplications. In this analysis, intra and inter-location variances arecombined to estimate the standard error of yield from transgenic plantsand control plants. Relative mean comparisons are used to indicatestatistically significant yield improvements.

C. Screening for Water Use Efficiency

Many transgenic crop plants of this invention exhibit improved yieldresulting from improved water use efficiency and/or drought tolerance.

A greenhouse screen for transgenic corn plants for water use efficiencymeasures changes in plant growth rate, e.g., at least a 10% improvement,in height and biomass during a vegetative drought treatment, as comparedto control plants. The hydration status of the shoot tissues followingthe drought is also measured. Shoot Initial Height (SIH) is plant heightafter 3 weeks of growth under optimum conditions. Shoot Wilt Height(SWH) is plant height at the end of a 6 day drought. Time courseexperiments have shown that at about 3 days of drought, wild type plantsbasically stop growing and begin to wilt. Thus a transgenic plant withimproved water use efficiency will continue to grow (probably to alesser extent than with water) and thereby end up as a significantlytaller plant at the end of a drought experiment. Shoot Wilt Mass (SWM)is the amount of wet and dry matter in the shoot (plant separated fromroot ball at the soil line) at the end of the drought; SDM is measureafter 2 to 3 weeks in a drying chamber. Shoot Turgid mass (STM) is theSWM plus the mass of the water that is transported into plant tissues in3 days of soaking in 40 degree C. water in the dark. Experiments showthat most of the water is pulled up in 24 hours but it takes 2 more daysbefore additional increase becomes insignificant. STM-SWM is indicativeof water use efficiency in plants where recovery from stress is moreimportant than stress tolerance per se. Relative water content (RWC) isa measurement of how much (%) of the plant is water at harvest.RWC=(SWM−SDM)/(STM−SDM)*100. Fully watered corn plants are about 98%RWC. Typically, in a wilt screen the plants are about 60% RWC. Plantswith higher RWC at the end of a drought are considered to be healthierplants and more fit for post-drought recovery and growth.Relative Growth Rate (RGR) is calculated for each shoot using theformula RGR=(SWH−SIH)/((SWH+SIH)/2)*100

D. Screening for Growth Under Cold Stress

Many transgenic crop plants of this invention exhibit improved growthunder cold stress, e.g., in a cold germination assay, in a cold shockassay, in an early seedling growth assay and in root-shoot biomassassay.

In a cold germination assay transgenic seeds from transgenic plants,e.g., R2 inbred seeds or F1 hybrid seeds, seeds of two types of controlplants, e.g., negative segregants from the transgenic event or wildtype, non-transgenic seeds of the transformed genotype, are treated withfungicide. A useful fungicide such as Captan fungicide (available fromArvesta Corp as MAESTRO® 80DF Fungicide) is applied at the rate of 0.43mL Captan per 45 g of corn seeds which are dried to providefungicide-coated seeds.

In a useful cold screen for transgenic corn seeds ten seeds pertransgenic event are placed on filter paper (e.g., Whatman No. 1) in thelid of a Petri dish with 5 ml of water. A closed Petri dish is placed ina growth chamber set at 11 degrees C. for inbred corn seed or 9.5degrees C. for hybrid corn seed. 2 ml of water is added on day 3 and day10. Seeds are considered germinated if the emerged radical size is 1 cm.Cold seeds are scored every 2 days from day 10 up to day 30. Tissuesamples are collected at random on the last day of the experiment forconfirmation of RNA expression. A germination index (GI) is calculatedasGI=(Σ([T+1−n _(i)]*[P _(i)-P _(i-1)]))/Twhere “T” is the number of days for the experiment, “n” is the number ofdays after start, “i” is number of times germination is countedincluding the current day, “P” is the percentage of seed germinatedduring any given rating. Statistical differences are calculated betweenpositive and wild type control.

In a cold shock assay, seeds are planted in potting media and placed ina growth chamber set at 23 degrees C., relative humidity of 65% with 12hour day and night photoperiod (300 uE/m2-min). Planted seeds arewatered for 20 minute every other day by sub-irrigation and flats arerotated every third day. On day 10 after planting the transgenicpositive and wild type control plants are positioned in flats in analternating pattern. Chlorophyll fluorescence of plants is measured onthe tenth day during the dark period of growth by using a Walz PAM-2000portable fluorometer following manufacturer's instructions. Afterchlorophyll measurements, leaf samples from each event are collected forconfirming the expression of recombinant DNA. The plants are thenexposed to temperatures of 5 degrees C. for 4 days. On the fourth daychlorophyll fluorescence is measured and plants are restored to a 23degrees C. environment for recovery over 3 days. During the recoveryperiod the length of the V3 leaf is measured on the first and thirddays. After two days of recovery V2 leaf damage is determined visuallyby estimating percent of green V2 leaf. Statistical differences in V3leaf growth, V2 leaf necrosis and fluorescence during pre-shock and coldshock can be used for estimation of cold shock damage on corn plants.

In an early seedling growth assay three sets of seeds are assayed. Thefirst set is a group of transgenic seeds from transgenic plants; thesecond set is negative segregants of the transgenic seed; and the thirdseed set is seed from two cold tolerant and two cold sensitive wild-typecontrols. All seeds are treated with a fungicide as indicated above.Seeds are grown in germination paper (12 inch×18 inch pieces of AnchorPaper #SD7606), wetted in a solution of 0.5% KNO₃ and 0.1% Thyram. Foreach paper fifteen seeds are placed on the line evenly spaced such thatthe radicals will grow toward the same edge. The wet paper is rolled upevenly and tight enough to hold the seeds in place. The roll is securedinto place with two large paper clips, one at the top and one at thebottom. The rolls are incubated in a growth chamber at 23 degrees C. forthree days in a randomized complete block design within an appropriatecontainer. The chamber is set for 65% humidity with no light cycle. Forthe cold stress treatment the rolls are then incubated in a growthchamber at 12 degrees C. for fourteen days. The chamber is set for 65%humidity with no light cycle. For the warm treatment the rolls areincubated at 23 degrees C. for an additional two days. After thetreatment the germination papers are unrolled and the seeds that did notgerminate are discarded. The lengths of the radical and coleoptile foreach seed are measured. A coleoptile sample is collected from sixindividual kernels of each entry for confirming the expression ofrecombinant DNA. Statistical differences in the length of radical andshoot during pre-shock and cold shock are used for an estimation of theeffect of the cold treatment on corn plants. The analysis is conductedindependently for the warm and cold treatments.

In a root-shoot biomass assay two sets of seeds are used. The first setis transgenic seeds with recombinant DNA, e.g., R2 inbred seeds or F1hybrid seeds; the second seed set is non-transgenic, wild type negativecontrol made from the same genotypes as the transgenic seeds. All seedsare treated with a fungicide as indicated above. The seeds are plantedin potting media in pots arranged in a randomized complete block designwith 6 replications. Pots are watered as and when needed by fillingwater up to the brim of the pot. Plants are grown in a greenhouse to aV6 stage or approximately for 28 days. Greenhouse lights are turned onafter emergence of seedlings with 14 hours of light 10 hours of dark.Plants are fertilized twice each week with water-soluble fertilizercontaining 200-ppm nitrogen. For measurement of root and shoot dryweight, two pots are separated carefully to remove adhering sand bywashing with water. Washed roots are cut at the first node. The rootsare placed in a paper bag after squeezing excess water, folded once andstapled. The shoots are then folded up to a convenient size(approximately 15 cm), placed in a paper bag. Bags are placed over awire shelve to facilitate drying in a ventilated room maintained at 120degrees F. to a moisture content of about 13% then weighed to determinedried root and shoot biomass.

E. Screen for Enhanced Oil, Starch, or Protein Levels in Plant Seeds

Oil concentrations are determined in kernels by Near InfraredTransmittance (NIT) from inbred and from hybrid lines. Data are alsoobtained for protein and starch content from this measurement.

Inbred Kernel Oil Screen

The primary transformants are selfed to produce R1 seed which is plantedto segregating seed. An untransformed control line is planted everysixth row. All plants are self-pollinated. A molecular assay isconducted to determine zygosity of the transgene in each plant. Ears areharvested at maturity, and well-filled ears are chosen for proximateanalysis. Proximate analysis is conducted on up to 5 homozygous ears. If5 good homozygous ears are not available, then hemizygous ears will beused to obtain 5 good transgene-positive ears. Statistical analysis isconducted to determine whether proximate values for transgenic eventsare different from controls. Events with an increase in oil with ap-value of less than or equal to 0.1 are termed “putative leads.” Kernelcomposition is confirmed in an inbred confirmation nursery which isconducted with selected events, and is run with a design similar to thatof the Gen2 nursery. A “confirmed lead event” demonstrates an increasein oil with a p-value of less than or equal to 0.1 in two nurseries.

Hybrid Kernel Oil Screen

Grain samples from the multilocation hybrid yield trials are collectedat the time of harvest and are analyzed by NIT. Controls are negativesegregants, untransformed controls, or pollinators. Data from 3 to 12locations are pooled for the statistical analysis. Putative leads haveincreased oil with a p-value of less than or equal to 0.1.

The various aspects of the invention are illustrated by means of thefollowing examples which are in no way intended to limit the full breathand scope of claims.

Example 1. Identification of Recombinant DNA that Confers ImprovedTrait(s) to Plants

A. Expression Constructs for Arabidopsis Plant Transformation

Each gene of interest was amplified from a genomic or cDNA library usingprimers specific to sequences upstream and downstream of the codingregion. Transformation vectors were prepared to constitutivelytranscribe DNA in either sense orientation (for enhanced proteinexpression) or anti-sense orientation (for endogenous gene suppression)under the control of an enhanced Cauliflower Mosaic Virus 35S promoter(U.S. Pat. No. 5,359,142) directly or indirectly (Moore, et al., PNAS95:376-381, 1998; Guyer, et al., Genetics 149: 633-639, 1998;International patent application NO. PCT/EP98/07577). The transformationvectors also contain a bar gene as a selectable marker for resistance toglufosinate herbicide. The transformation of Arabidopsis plants wascarried out using the vacuum infiltration method known in the art(Bethtold, et al., Methods Mol. Biol. 82:259-66, 1998). Seeds harvestedfrom the plants, named as T1 seeds, were subsequently grown in aglufosinate-containing selective medium to select for plants which wereactually transformed and which produced T2 transgenic seed.

B. Soil Drought Tolerance Screen

This example describes a soil drought tolerance screen to identifyArabidopsis plants transformed with recombinant DNA that wilt lessrapidly and/or produce higher seed yield when grown in soil underdrought conditions

T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts®Company, USA). Humidity domes were added to each flat and flats wereassigned locations and placed in climate-controlled growth chambers.Plants were grown under a temperature regime of 22° C. at day and 20° C.at night, with a photoperiod of 16 hours and average light intensity of170 μmol/m²/s. After the first true leaves appeared, humidity domes wereremoved. The plants were sprayed with glufosinate herbicide and put backin the growth chamber for 3 additional days. Flats were watered for 1hour the week following the herbicide treatment. Watering was continuedevery seven days until the flower bud primordia became apparent, atwhich time plants were watered for the last time.

To identify drought tolerant plants, plants were evaluated for wiltingresponse and seed yield. Beginning ten days after the last watering,plants were examined daily until 4 plants/line had wilted. In the nextsix days, plants were monitored for wilting response. Five droughtscores were assigned according to the visual inspection of thephenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severewilting, and 5 for dead. A score of 3 or higher was considered aswilted.

At the end of this assay, seed yield measured as seed weight per plantunder the drought condition was characterized for the transgenic plantsand their controls and analyzed as a quantitative response according toexample 1M.

Two approaches were used for statistical analysis on the wiltingresponse. First, the risk score was analyzed for wilting phenotype andtreated as a qualitative response according to the example 1L.Alternatively, the survival analysis was carried out in which theproportions of wilted and non-wilted transgenic and control plants werecompared over each of the six days under scoring and an overall log ranktest was performed to compare the two survival curves using S-PLUSstatistical software (S-PLUS 6, Guide to statistics, Insightful,Seattle, Wash., USA). Table 4 provides a list of recombinant DNAconstructs that improve drought tolerance in transgenic plants.

TABLE 4 Survival Anaysis of Wilt Response Seed Weight/ wilt response PepRisk score plant diff SEQ RS p- p- time to p- ID Construct_id GeneOrientation mean value c delta value c wilting value c 319 10139 CGPG101ANTI-SENSE 0.115 0.024 S −0.123 0.804 / −0.01 0.469 / 320 11410 CGPG103SENSE 0.226 0.003 S −0.366 0.926 / 0 1 / 321 11604 CGPG48 ANTI-SENSE0.25 0.034 S 0.257 0.01 S −0.24 0.38 / 322 12368 CGPG1006 SENSE 0.1480.044 S 0.359 0.02 S −0.06 0.764 / 323 13502 CGPG1354 SENSE 0.431 0 S0.624 0 S 1.34 0.366 / 324 13745 CGPG1576 ANTI-SENSE −0.021 0.711 /0.664 0 S −0.29 0.453 / 325 13821 CGPG1569 SENSE 0.135 0.025 S 0.1280.402 / 0.24 0.972 / 326 14240 CGPG1697 SENSE 0.377 0.002 S −1.305 0.991/ 0 1 / 327 14718 CGPG1082 SENSE 0.168 0.001 S 0.135 0.351 / 0.25 0.208/ 328 17022 CGPG1774 SENSE 0.06 0.124 T 0.563 0.043 S 0 0.961 / 32917924 CGPG2882 SENSE −0.093 0.914 / 0.288 0.021 S 0.09 0.935 / 330 18259CGPG3368 SENSE 0.07 0.28 / 0.391 0.058 T −0.27 0.591 / 331 19171CGPG2952 SENSE 0.227 0.005 S 0.846 0.001 S 0.12 0.543 / 332 19201CGPG2332 SENSE 0.124 0.027 S −0.435 0.785 / 0.35 0.256 / 333 19317CGPG3662 SENSE 0.338 0 S −0.071 0.61 / 0.63 0.106 T 334 70417 CGPG3427SENSE 0.253 0.016 S −1.424 0.984 / 0 1 / 315 70427 CGPG3067 SENSE −0.0330.818 / 1.004 0.002 S 0.01 0.977 / 335 70467 CGPG3785 SENSE 0.127 0.023S −0.448 0.946 / 0.71 0.046 S 336 70806 CGPG712 SENSE 0.246 0.046 S0.174 0.276 / 0.14 0.07 T 337 70818 CGPG479 SENSE 0.07 0.115 T 0.5580.009 S 0.61 0.283 / 338 70820 CGPG655 SENSE 0.172 0.048 S 0.036 0.441 /0.26 0.554 / 339 70919 CGPG4029 SENSE 0.167 0.009 S −0.565 0.904 / 0.310.508 / 340 71623 CGPG4696 SENSE 0.158 0.047 S 0.421 0.04 S 0 1 / 39071633 CGPG857 SENSE 0.121 0.017 S −0.823 0.967 / 0.45 0.139 T 341 71662CGPG4679 SENSE 0.063 0.013 S −0.037 0.631 / 0.16 0.957 / 342 71693CGPG4652 SENSE 0.074 0.042 S 0.246 0.06 T 0.34 0.616 / 316 71811CGPG4426 SENSE 0.359 0.015 S −0.729 0.903 / 0.15 0.822 / 318 72081CGPG5279 SENSE 0.269 0.005 S −0.372 0.987 / 0.17 0.404 / 343 72384CGPG4639 SENSE 0.133 0.018 S 0.62 0.002 S 0.17 0.359 / 344 72439CGPG5075 SENSE 0.175 0.013 S −0.035 0.604 / 0.23 0.244 / 374 72456CGPG4745 SENSE 0.53 0.01 S −0.737 0.979 / −0.08 0.823 / 345 72619CGPG4835 SENSE 0.178 0.039 S 0.219 0.072 T 0.96 0.691 / 346 72624CGPG4842 SENSE 0.163 0.021 S 0.356 0.051 T 0.3 0.375 / 347 72715CGPG5521 SENSE 0.082 0.1 T 0.767 0.002 S 0.12 0.628 / 348 72754 CGPG5548SENSE 0.131 0.031 S −0.365 0.974 / 0 0.923 / 349 72819 CGPG4989 SENSE0.094 0.026 S 0.362 0.069 T 0.03 0.83 / 350 75516 CGPG7689 SENSE 0.0670.066 T 0.965 0.001 S 0.24 0.464 / 351 75701 CGPG7856 SENSE 0.147 0.006S 0.986 0.015 S 0.15 0.448 / 317 73463 CGPG6384 SENSE 0.174 0.048 S−1.359 0.959 / 0.09 0.984 / 454 70354 CGPG3995 SENSE −0.005 0.563 /0.444 0.002 S 10.29 0.9 / 397 71840 CGPG4353 SENSE 0.142 0.007 S −0.2120.859 / 9.29 0.99 / 506 72902 CGPG5597 SENSE 0.009 0.162 T 0.034 0.2 T 51 / 437 73549 CGPG6460 SENSE 0.119 0.037 S −0.774 0.949 / 6.26 0.25 /302 73586 CGPG6471 SENSE 0.003 0.451 / 0.588 0.002 S 6.34 0.723 / 44274587 CGPG6774 SENSE 0.262 0.001 S −0.117 0.574 / 7.49 0.041 S 388 74652CGPG6168 SENSE 0.475 0 S −0.766 0.92 / 7.48 0 S S: represents that thetransgenic plants showed statistically significant trait improvement ascompared to the reference (p < 0.05, p value, of the delta of aquantitative response or of the risk score of a qualitative response, isthe probability that the observed difference between the transgenicplants and the reference occur by chance) T: represents that thetransgenic plants showed a trend of trait improvement as compared to thereference with p < 0.2 /: represents the transgenic plants didn't showany alteration or had unfavorable change in traits examined as comparedto the reference in the current dataset.

C. Heat Stress Tolerance Screen

Under high temperatures, Arabidopsis seedlings become chlorotic and rootgrowth is inhibited. This example sets forth the heat stress tolerancescreen to identify Arabidopsis plants transformed with the gene ofinterest that are more resistant to heat stress based on primarily theirseedling weight and root growth under high temperature.

T2 seeds were plated on ½×MS salts, 1/% phytagel, with 10 μg/ml BASTA (7per plate with 2 control seeds; 9 seeds total per plate). Plates wereplaced at 4° C. for 3 days to stratify seeds. Plates were then incubatedat room temperature for 3 hours and then held vertically for 11additional days at temperature of 34° C. at day and 20° C. at night.Photoperiod was 16 h. Average light intensity was ˜140 μmol/m²/s. After14 days of growth, plants were scored for glufosinate resistance, rootlength, final growth stage, visual color, and seedling fresh weight. Aphotograph of the whole plate was taken on day 14.

The seedling weight and root length were analyzed as quantitativeresponses according to example 1M. The final grow stage at day 14 wasscored as success if 50% of the plants had reached 3 rosette leaves andsize of leaves are greater than 1 mm (Boyes, et al., (2001) The PlantCell 13, 1499-1510). The growth stage data was analyzed as a qualitativeresponse according to example 1L. Table 5 provides a list of recombinantDNA constructs that improve heat tolerance in transgenic plants.

TABLE 5 Pep Growth stage Root Length Seedling Weight SEQ RS p- p- p- IDConstruct_id Gene Orientation mean value c delta value c delta value c354 19542 CGPG3069 SENSE 0.842 0.021 S 0.296 0.023 S 1.616 0 S 355 19618CGPG3574 SENSE 1.072 0.005 S 0.328 0.006 S 1.569 0 S 356 19649 CGPG3140SENSE 0.72 0.042 S 0.243 0.054 T 1.665 0 S 357 19745 CGPG3973 SENSE 0.650.016 S 0.19 0.021 S 1.505 0 S 358 19768 CGPG4096 SENSE 1.06 0.035 S0.17 0.034 S 1.171 0 S 436 19771 CGPG4011 SENSE 0.822 0.023 S 0.2010.001 S 1.43 0 S 359 19772 CGPG3939 SENSE 0.247 0.014 S 0.151 0.014 S1.344 0 S 360 19779 CGPG4113 SENSE 0.605 0.051 T 0.181 0.003 S 1.436 0 S361 19833 CGPG4074 SENSE 0.965 0.026 S 0.266 0.007 S 1.01 0 S 362 19862CGPG3961 SENSE 0.341 0.119 T 0.132 0.007 S 1.22 0 S 363 19879 CGPG4009SENSE 0.734 0.002 S 0.22 0.001 S 1.499 0 S 364 70445 CGPG3728 SENSE0.413 0.062 T 0.148 0.114 T 1.249 0 S 365 70738 CGPG3195 SENSE 0.6870.055 T 0.205 0.041 S 1.261 0 S 366 71437 CGPG4043 SENSE 0.094 0.198 T0.092 0.064 T 1.301 0 S 367 71572 CGPG4520 SENSE 0.938 0.052 T 0.441 0 S1.633 0 S 368 71617 CGPG1227 SENSE 0.809 0.012 S 0.143 0.029 S 1.050.003 S 408 72085 CGPG5228 SENSE 1.234 0.02 S 0.192 0.043 S 1.162 0 S369 72532 CGPG4780 SENSE 1.028 0.022 S 0.198 0.052 T 1.043 0.001 S 40972744 CGPG5563 SENSE 0.17 0.146 T 0.04 0.359 / 0.827 0.004 S 370 72757CGPG5572 SENSE 1.82 0.004 S 0.14 0.091 T 1.121 0 S 407 72771 CGPG2166SENSE 1.776 0.001 S 0.36 0 S 1.428 0 S 444 72967 CGPG5742 SENSE 0.2730.063 T 0.147 0.102 T 1.03 0 S 410 73039 CGPG810 SENSE −0.048 0.774 /−0.135 0.957 / 0.59 0.022 S 411 73054 CGPG5754 SENSE 0.055 0.312 / 0.2360.001 S 1.434 0 S 508 73055 CGPG5768 SENSE 0.154 0.123 T 0.269 0 S 1.5240 S 371 73412 CGPG6448 SENSE 0.187 0.118 T 0.134 0.06 T 1.181 0 S 41273501 CGPG6456 SENSE 1.6 0.003 S 0.081 0.136 T 1.119 0 S 352 73515CGPG6473 SENSE −0.037 0.758 / −0.024 0.604 / 0.694 0.008 S 437 73549CGPG6460 SENSE 2.612 0 S 0.199 0.017 S 1.432 0 S 372 74102 CGPG6550SENSE 0.34 0.035 S 0.268 0.002 S 1.355 0 S 509 74103 CGPG6558 SENSE−0.013 1 / −0.021 0.608 / 0.86 0 S 353 74684 CGPG6360 SENSE 0.44 0.018 S0.254 0.002 S 1.383 0 S 512 19703 CGPG4172 SENSE 0.211 0.079 T 0.0590.301 / 1.262 0 S 273 70423 CGPG3165 SENSE 1.456 0 S 0.418 0 S 1.912 0 S491 70469 CGPG3791 SENSE 0.143 0.28 / 0.028 0.349 / 1.001 0.001 S 43470932 CGPG4089 SENSE 0.354 0.1 T 0.03 0.419 / 1.254 0 S 419 71134CGPG817 SENSE 0.522 0.106 T 0.077 0.248 / 1.225 0 S 466 71726 CGPG3894SENSE 0.196 0.225 / 0.082 0.2 T 1.243 0 S 505 72463 CGPG4760 SENSE 1.1820.003 S 0.202 0.026 / 1.702 0 S 445 72961 CGPG5591 SENSE 1.195 0.013 S0.106 0.105 T 1.084 0 S 306 74136 CGPG6632 SENSE 0.886 0.012 S 0.3060.009 S 1.42 0 S 504 74259 CGPG5343 SENSE 1.044 0.023 S 0.081 0.187 T1.274 0 S 310 74318 CGPG5826 SENSE 0.407 0.075 T 0.116 0.052 T 1.118 0 S479 74344 CGPG5929 SENSE 1.256 0.018 S 0.118 0.125 T 1.275 0 S 533 74462CGPG6668 SENSE 0.846 0.022 S 0.26 0.011 S 1.425 0 S 313 74512 CGPG32SENSE 0.241 0.118 T 0.146 0.044 S 1.062 0 S 442 74587 CGPG6774 SENSE 0 // 0.084 0.054 T 1.046 0 S S: represents the transgenic plants showedstatistically significant trait improvement as compared to the reference(p < 0.05) T: represents the transgenic plants showed a trend of traitimprovement as compared to the reference with p < 0.2 /: represents datapoints not determined or the transgenic plants didn't show anyalteration or had unfavorable change in traits examined as compared tothe reference in the current dataset

D. Salt Stress Tolerance Screen

This example sets forth the high salinity stress screen to identifyArabidopsis plants transformed with the gene of interest that aretolerant to high levels of salt based on their rate of development, rootgrowth and chlorophyll accumulation under high salt conditions.

T2 seeds were plated on glufosinate selection plates containing 90 mMNaCl and grown under standard light and temperature conditions. Allseedlings used in the experiment were grown at a temperature of 22° C.at day and 20° C. at night, a 16-hour photoperiod, an average lightintensity of approximately 120 umol/m². On day 11, plants were measuredfor primary root length. After 3 more days of growth (day 14), plantswere scored for transgenic status, primary root length, growth stage,visual color, and the seedlings were pooled for fresh weightmeasurement. A photograph of the whole plate was also taken on day 14.

The seedling weight and root length were analyzed as quantitativeresponses according to example 1M. The final growth stage at day 14 wasscored as success if 50% of the plants reached 3 rosette leaves and sizeof leaves are greater than 1 mm (Boyes, D. C., et al., (2001), The PlantCell 13, 1499/1510). The growth stage data was analyzed as a qualitativeresponse according to example 1L. Table 6 provides a list of recombinantDNA constructs that improve high salinity tolerance in transgenic plants

TABLE 6 Root Length Root Length Seedling Weight Pep Growth Stage at day11 at day 14 at day 14 SEQ RS p- p- p- p- ID Construct id GeneOrientation mean value c delta value c delta value c delta vallue c 51219703 CGPG4172 SENSE 1.124 0.139 T 0.198 0.021 S 0.072 0.116 T 0.5820.023 S 513 19946 CGPG4097 SENSE 1.201 0.072 T 0.02 0.89 / 0.069 0.573 /0.443 0.266 / 514 19980 CGPG3914 SENSE 0.904 0.146 T 0.101 0.259 / 0.1440.058 T 0.706 0.016 S 515 70435 CGPG3701 SENSE 1.363 0.031 S −0.1180.228 / 0.161 0.038 S 0.053 0.697 / 516 71114 CGPG1657 SENSE 0.138 0.399/ 0.245 0.009 S 0.187 0.025 S 0.472 0.069 T 517 72451 CGPG4733 SENSE2.226 0.02 S 0.186 0.006 S 0.069 0.23 / 0.466 0.011 S 443 72453 CGPG4735SENSE 1.539 0.031 S 0.232 0.002 S 0.216 0 S 0.737 0.001 S 505 72463CGPG4760 SENSE 3.026 0.002 S 0.119 0.269 / 0.202 0.073 T 1.26 0 S 39272519 CGPG4749 SENSE 0.598 0.041 S 0.126 0.055 T 0.172 0.003 S 0.6350.008 S 506 72902 CGPG5597 SENSE 1.418 0.039 S 0.114 0.286 / 0.226 0.078T 0.426 0.091 T 448 72916 CGPG1814 SENSE 0.682 0.181 T 0.07 0.532 /0.201 0.046 S 0.387 0.079 T 510 72921 CGPG5781 SENSE 1.977 0.029 S 0.1630.176 T 0.211 0.073 T 0.663 0.006 S 518 72947 CGPG5607 SENSE 1.505 0.028S 0.024 0.899 / 0.204 0.022 S 0.466 0.216 / 445 72961 CGPG5591 SENSE1.879 0.007 S 0.228 0.122 T 0.229 0.003 S 0.817 0.04 S 444 72967CGPG5742 SENSE 2.427 0.004 S 0.386 0.009 S 0.369 0.001 S 1.254 0 S 51172968 CGPG5772 SENSE 1.531 0.055 T 0.302 0.06 T 0.209 0.073 T 0.7610.003 S 449 72969 CGPG5789 SENSE 0.67 0.078 T 0.029 0.789 / 0.239 0.008S 0.603 0.013 S 519 73012 CGPG5786 SENSE 2.371 0.001 S 0.366 0 S 0.342 0S 1.08 0 S 520 73022 CGPG5622 SENSE 1.408 0.036 S 0.22 0.056 T 0.347 0 S0.492 0.03 S 508 73055 CGPG5768 SENSE 3.291 0.001 S 0.369 0.087 T 0.4170.005 S 1.096 0.005 S 446 73070 CGPG5627 SENSE 2.755 0.005 S 0.188 0.392/ 0.275 0.011 S 0.685 0.025 S 447 73475 CGPG6385 SENSE 1.11 0.059 T0.127 0.361 / 0.216 0.001 S 0.474 0.06 T 521 73488 CGPG6394 SENSE 2.3730.013 S 0.314 0.006 S 0.262 0.002 S 1.126 0.005 S 522 73901 CGPG5237SENSE 1.141 0.078 T 0.207 0.197 T 0.202 0.097 T 0.639 0.03 S 523 73964CGPG5804 SENSE 1.235 0.043 S 0.428 0.008 S 0.317 0.022 S 0.955 0.002 S524 74019 CGPG5706 SENSE 0.105 0.168 T 0.074 0.649 / 0.171 0.106 T 0.7730.011 S 525 74022 CGPG5724 SENSE 0.033 0.327 / −0.065 0.616 / 0.1720.032 S 0.484 0.068 T 509 74103 CGPG6558 SENSE 1.225 0.074 T 0.26 0.042S 0.267 0.004 S 0.543 0.042 S 526 74114 CGPG6551 SENSE 3.627 0 S 0.2650.119 T 0.26 0 S 0.561 0.063 T 504 74259 CGPG5343 SENSE 2.802 0.003 S0.249 0.098 T 0.256 0.037 S 0.995 0 S 527 74262 CGPG5353 SENSE 0.2250.319 / 0.238 0.062 T 0.247 0 S 0.629 0.006 S 528 74292 CGPG5367 SENSE0.327 0.199 T 0.16 0.067 T 0.105 0.166 T 0.565 0.013 S 529 74302CGPG5384 SENSE 1.246 0.016 S 0.296 0.004 S 0.25 0 S 0.705 0.004 S 53074325 CGPG5898 SENSE 1.596 0.021 S −0.035 0.76 / 0.094 0.106 T 0.6850.004 S 531 74429 CGPG6689 SENSE 1.796 0.008 S 0.298 0.037 S 0.207 0.006S 0.496 0.029 S 532 74440 CGPG6682 SENSE 0.223 0.334 / 0.43 0.007 S0.272 0.01 S 0.744 0.017 S 450 74449 CGPG6659 SENSE 0.693 0.19 T 0.2040.104 T 0.205 0.022 S 0.451 0.095 T 533 74462 CGPG6668 SENSE 2.14 0.028S 0.244 0.038 S 0.239 0.001 S 0.64 0.013 S 534 74465 CGPG6692 SENSE1.245 0.016 S 0.35 0.01 S 0.215 0.001 S 0.575 0.043 S 535 74474 CGPG6669SENSE 3.312 0.002 S 0.233 0.083 T 0.338 0.003 S 0.589 0.044 S 536 74505CGPG6783 SENSE 1.731 0.043 S 0.272 0.007 S 0.208 0.01 S 0.493 0.009 S537 74507 CGPG6799 SENSE 2.32 0.009 S 0.056 0.567 / 0.227 0.035 S 0.4760.126 T 538 74562 CGPG6764 SENSE 1.405 0.038 S −0.052 0.776 / 0.2150.014 S 0.104 0.766 / 507 74572 CGPG6640 SENSE 1.425 0.025 S 0.201 0.009S 0.267 0.001 S 1.184 0 S 270 14324 CGPG1560 SENSE 1.708 0.017 S 0.3070.01 S 0.408 0 S 0.995 0.002 S 358 19768 CGPG4096 SENSE 1.496 0.061 T0.163 0.187 T 0.129 0.013 S 0.66 0.002 S 436 19771 CGPG4011 SENSE 1.6660.051 T 0.319 0.005 S 0.201 0.028 S 0.68 0.031 S 363 19879 CGPG4009SENSE 2.117 0.029 S 0.16 0.139 T 0.091 0.202 / 0.545 0.028 S 347 72715CGPG5521 SENSE 1.078 0.002 S 0.207 0.141 T 0.176 0.014 S 0.519 0.015 S407 72771 CGPG2166 SENSE 0.564 0.256 / −0.019 0.857 / 0.206 0.013 S0.306 0.05 S 440 72903 CGPG5584 SENSE 0.645 0.196 T −0.031 0.569 / 0.0970.389 / 0.482 0.015 S 411 73054 CGPG5754 SENSE 1.52 0.035 S 0.198 0.089T 0.098 0.187 T 0.696 0.004 S 476 74107 CGPG6590 SENSE 0.772 0.036 S0.411 0 S 0.431 0.001 S 1.579 0 S 478 74131 CGPG6592 SENSE 1.747 0.044 S0.316 0.004 S 0.115 0.01 S 0.663 0.001 S 442 74587 CGPG6774 SENSE 1.7540.01 S 0.121 0.243 / 0.194 0.001 S 0.681 0 S S: represents thetransgenic plants showed statistically significant trait improvement ascompared to the reference (p < 0.05) T: represents the transgenic plantsshowed a trend of trait improvement as compared to the reference with p< 0.2 /: represents the transgenic plants didn't show any alteration orhad unfavorable change in traits examined as compared to the referencein the current dataset

E. Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance Screen

There are numerous factors, which can influence seed germination andsubsequent seedling growth, one being the availability of water. Genes,which can directly affect the success rate of germination and earlyseedling growth, are potentially useful agronomic traits for improvingthe germination and growth of crop plants under drought stress. In thisassay, PEG was used to induce osmotic stress on germinating transgeniclines of Arabidopsis thaliana seeds in order to screen for osmoticallyresistant seed lines.

T2 seeds were plated on BASTA selection plates containing 3% PEG andgrown under standard light and temperature conditions. Seeds were platedon each plate containing 3% PEG, ½×MS salts, 1% phytagel, and 10 μg/mlglufosinate. Plates were placed at 4° C. for 3 days to stratify seeds.On day 11, plants were measured for primary root length. After 3 moredays of growth, i.e., at day 14, plants were scored for transgenicstatus, primary root length, growth stage, visual color, and theseedlings were pooled for fresh weight measurement. A photograph of thewhole plate was taken on day 14.

Seedling weight and root length were analyzed as quantitative responsesaccording to example 1M. The final growth stage at day 14 was scored assuccess or failure based on whether the plants reached 3 rosette leavesand size of leaves are greater than 1 mm. The growth stage data wasanalyzed as a qualitative response according to example 1L. Table 7provides a list of recombinant DNA constructs that improve osmoticstress tolerance in transgenic plants.

TABLE 7 Root Length Root Length Seedling Weight Pep Growth Stage at day11 at day 14 at day 14 SEQ RS p- p- p- p- ID Gene Construct_idOrientation mean value c delta value c delta value c delta value c 41319707 CGPG4179 SENSE 2.653 0.019 S 0.074 0.58 T −0.031 0.81 / 0.4270.063 T 414 19951 CGPG3941 SENSE 1.432 0.134 T 0.017 0.864 T −0.08 0.28/ 0.476 0.054 T 415 19967 CGPG4032 SENSE 2.691 0.014 S 0.01 0.934 T 0.190.039 S 0.537 0.056 T 416 70543 CGPG3815 SENSE 1.735 0.077 T 0.084 0.561T 0.323 0.007 S 0.676 0.006 S 402 70681 CGPG4584 SENSE 2.528 0.006 S−0.065 0.682 / −0.179 0.05 / 0.374 0.146 T 417 70707 CGPG1273 ANTI-SENSE1.42 0.095 T 0.248 0.006 S 0.3 0.002 S 0.331 0.007 S 418 70719 CGPG1712ANTI-SENSE 1.43 0.106 T −0.007 0.968 / 0.101 0.255 T 0.155 0.349 T 41971134 CGPG817 SENSE 1.478 0.13 T 0.119 0.15 T 0.035 0.684 T 0.198 0.277T 420 71146 CGPG2928 SENSE 2.624 0.016 S 0.227 0.101 T 0.242 0.1 T 0.2780.296 T 405 71508 CGPG1541 SENSE 3.153 0.001 S 0.446 0.054 T 0.384 0.048S 0.782 0.003 S 421 71660 CGPG4690 SENSE 2.893 0.005 S −0.024 0.795 /0.225 0.074 T 0.165 0.329 T 403 71663 CGPG4638 SENSE 0.116 0.444 T−0.061 0.536 / 0.358 0.041 S 0.058 0.702 T 439 71928 CGPG1617 SENSE2.076 0.041 S 0.353 0.013 S 0.289 0.035 S 0.531 0.035 S 318 72081CGPG5279 SENSE 1.262 0.138 T 0.174 0.243 T 0.2 0.197 T −0.105 0.714 /408 72085 CGPG5228 SENSE 4 0 S 0.22 0.046 S 0.371 0.004 S 0.929 0 S 42272086 CGPG5236 SENSE 2.589 0.022 S 0.281 0.033 S 0.136 0.314 T 0.320.043 S 423 72632 CGPG4852 SENSE 1.663 0.053 T 0.254 0.069 T 0.094 0.548T 0.471 0.015 S 424 72716 CGPG5529 SENSE 2.914 0.004 S 0.146 0.058 T0.007 0.925 T 0.582 0.016 S 425 72723 CGPG1848 SENSE 2.138 0.066 T 0.0430.728 T 0.2 0.068 T −0.326 0.225 / 409 72744 CGPG5563 SENSE 1.636 0.05 /0.195 0.151 T 0.059 0.751 T 0.539 0.005 S 404 72769 CGPG5573 SENSE 2.2070.055 T 0.086 0.464 T −0.134 0.162 / 0.389 0.086 T 407 72771 CGPG2166SENSE 2.569 0.021 S 0.169 0.221 T 0.192 0.038 S 0.56 0.018 S 440 72903CGPG5584 SENSE 2.161 0.061 T 0.035 0.856 T 0.245 0.162 T 0.06 0.871 T510 72921 CGPG5781 SENSE 2.249 0.025 S 0.034 0.788 T 0.225 0.077 T 0.3060.28 T 391 72948 CGPG5617 SENSE 2.267 0.005 S 0.054 0.495 T 0.117 0.019S 0.289 0.077 T 445 72961 CGPG5591 SENSE 1.19 0.14 T 0.037 0.76 T 0.1360.339 T 0.588 0.027 S 511 72968 CGPG5772 SENSE 3.142 0.007 S 0.178 0.072T −0.031 0.781 / 0.698 0.016 S 426 72987 CGPG1787 SENSE 2.055 0.078 T−0.058 0.64 / 0.097 0.144 T 0.325 0.054 T 438 72994 CGPG5803 SENSE 2.6740.013 S 0.245 0.124 T 0.07 0.657 T 0.599 0.035 S 441 73017 CGPG5733SENSE 4 0 S 0.343 0.09 T 0.462 0.002 S 0.996 0 S 410 73039 CGPG810 SENSE4 0 S 0.319 0.019 S 0.237 0.05 / 0.426 0.011 S 411 73054 CGPG5754 SENSE3.048 0.002 S 0.556 0.003 S 0.26 0.063 T 1.12 0.002 S 446 73070 CGPG5627SENSE 3.439 0.001 S 0.139 0.49 T 0.19 0.135 T 0.24 0.365 T 447 73475CGPG6385 SENSE 1.933 0.033 S 0.04 0.476 T 0.009 0.897 T 0.459 0.073 T412 73501 CGPG6456 SENSE 1.29 0.121 T 0.143 0.118 T 0.043 0.564 T 0.4330.013 S 427 74109 CGPG6606 SENSE 3.517 0 S 0.159 0.136 T 0.249 0.004 S0.333 0.025 S 428 74140 CGPG6569 SENSE 2.05 0.039 S 0.168 0.138 T 0.1960.086 T 0.71 0.012 S 429 74191 CGPG6597 SENSE 2.565 0.019 S 0.336 0.092T 0.199 0.112 T 0.54 0.02 S 406 74248 CGPG5476 SENSE 3.158 0.007 S 0.140.192 T 0.204 0.051 T 0.377 0.037 S 430 74265 CGPG5356 SENSE 2.208 0.023S 0.317 0.034 S 0.419 0.006 S 0.577 0.008 S 431 74369 CGPG6076 SENSE3.522 0 S 0.347 0.045 S 0.272 0.107 T 0.624 0.02 S 442 74587 CGPG6774SENSE 3.325 0.002 S 0.073 0.468 T 0.414 0.002 S 0.577 0.016 S 405 71508CGPG1541 SENSE 3.153 0.001 S 0.446 0.054 T 0.384 0.048 S 0.782 0.003 S439 71928 CGPG1617 SENSE 2.076 0.041 S 0.353 0.013 S 0.289 0.035 S 0.5310.035 S 422 72086 CGPG5236 SENSE 2.589 0.022 S 0.281 0.033 S 0.136 0.314/ 0.32 0.043 S 469 72455 CGPG4742 SENSE 1.367 0.15 T 0.026 0.764 / 0.0240.786 / 0.255 0.04 S 382 72466 CGPG4767 SENSE 0.735 0.101 T 0.068 0.333/ 0.249 0.006 S −0.341 0.278 / 373 72633 CGPG4853 SENSE 1.179 0.122 T0.227 0.009 S 0.097 0.065 T 0.442 0.013 S 370 72757 CGPG5572 SENSE 2.2720.017 S 0.125 0.361 / 0.11 0.317 / 0.425 0.047 S 472 72992 CGPG5777SENSE 2.233 0.056 T 0.176 0.261 / 0.116 0.34 / 0.511 0.019 S 438 72994CGPG5803 SENSE 2.674 0.013 S 0.245 0.124 T 0.07 0.657 / 0.599 0.035 S441 73017 CGPG5733 SENSE 4 0 S 0.343 0.09 T 0.462 0.002 S 0.996 0 S 41173054 CGPG5754 SENSE 3.048 0.002 S 0.556 0.003 S 0.26 0.063 T 1.12 0.002S 300 73507 CGPG6504 SENSE 0.343 0.334 / 0.347 0.007 S 0.261 0.032 S 0.30.122 T 352 73515 CGPG6473 SENSE 3.336 0.002 S 0.279 0.009 S 0.241 0.003S 0.328 0.05 S 428 74140 CGPG6569 SENSE 2.05 0.039 S 0.168 0.138 T 0.1960.086 T 0.71 0.012 S 430 74265 CGPG5356 SENSE 2.208 0.023 S 0.317 0.034S 0.419 0.006 S 0.577 0.008 S 431 74369 CGPG6076 SENSE 3.522 0 S 0.3470.045 S 0.272 0.107 T 0.624 0.02 S 422 74587 CGPG6774 SENSE 3.325 0.002S 0.073 0.468 / 0.414 0.002 S 0.577 0.016 S S: represents the transgenicplants showed statistically significant trait improvement as compared tothe reference (p < 0.05) T: represents the transgenic plants showed atrend of trait improvement compared to the reference with p < 0.2 /:represents the transgenic plants didn't show any alteration or hadunfavorable change in traits examined as compared to the reference inthe current dataset

F. Cold Shock Tolerance Screen

This example set forth a screen to identify Arabidopsis plantstransformed with the genes of interest that are more tolerant to coldstress subjected during day 8 to day 28 after seed planting. Duringthese crucial early stages, seedling growth and leaf area increase weremeasured to assess tolerance when Arabidopsis seedlings were exposed tolow temperatures. Using this screen, genetic alterations can be foundthat enable plants to germinate and grow better than wild type plantsunder sudden exposure to low temperatures.

Eleven seedlings from T2 seeds of each transgenic line plus one controlline were plated together on a plate containing ½× Gamborg Salts with0.8 Phytagel™, 1% Phytagel, and 0.3% Sucrose. Plates were then orientedhorizontally and stratified for three days at 4° C. At day three, plateswere removed from stratification and exposed to standard conditions (16hr photoperiod, 22° C. at day and 20° C. at night) until day 8. At dayeight, plates were removed from standard conditions and exposed to coldshock conditions (24 hr photoperiod, 8° C. at both day and night) untilthe final day of the assay, i.e., day 28. Rosette areas were measured atday 8 and day 28, which were analyzed as quantitative responsesaccording to example 1M. Table 8 provides a list of recombinantnucleotides that improve cold shock stress tolerance in plants.

TABLE 8 difference in rosette area rosette area rosette area between day28 Pep at day 8 at day 28 and day 8 SEQ p- p- p- ID Construct_id GeneOrientation delta value c delta value c delta value c 270 14324 CGPG1560SENSE 0.054 0.429 / 0.258 0.017 S −0.071 0.631 / 271 17484 CGPG2630SENSE −0.189 0.759 / 0.544 0.025 S 0.275 0.121 T 272 19109 CGPG1381ANTI-SENSE −0.016 0.523 / 0.541 0.008 S 0.818 0.014 S 273 70423 CGPG3165SENSE 0.316 0.012 S 0.521 0.022 S 0.89 0.018 S 274 70424 CGPG3180 SENSE0.474 0.003 S 0.695 0.003 S 0.693 0.043 S 275 70480 CGPG3833 SENSE−0.066 0.591 / 0.175 0.159 T 0.474 0.059 T 276 70509 CGPG2420 SENSE0.023 0.438 / 0.117 0.216 / 0.609 0.032 S 277 70647 CGPG4334 SENSE−0.508 0.894 / 0.604 0.049 S 0.895 0.047 S 278 70675 CGPG4519 SENSE 0.20.2 / 0.303 0.153 T 0.507 0.034 S 279 70829 CGPG518 SENSE −0.319 0.823 /0.804 0.002 S 1.082 0.002 S 280 70849 CGPG596 SENSE −0.039 0.564 / 0.6980.001 S 0.707 0.001 S 281 71627 CGPG1270 SENSE −0.146 0.748 / 0.349 0.05T 0.3 0.12 T 282 71934 CGPG2294 SENSE −0.068 0.796 / 0.757 0 S 0.922 0 S283 72615 CGPG4829 SENSE 0.477 0.007 S 0.834 0 S 0.979 0.001 S 286 73559CGPG6535 SENSE 0.143 0.093 T −0.265 0.878 / −0.344 0.821 / 287 74251CGPG5489 SENSE 0.377 0.021 S 0.439 0.045 S 0.45 0.07 T 389 70437CGPG3706 SENSE −0.273 0.916 / 0.147 0.165 T 0.682 0.034 S 402 70681CGPG4584 SENSE 0.352 0.155 T 0.252 0.261 / 0.269 0.328 / 403 71663CGPG4638 SENSE 0.358 0.013 S 0.032 0.423 / −0.031 0.585 / 404 72769CGPG5573 SENSE 0.381 0.049 S 0.881 0.006 S 1.102 0.005 S 407 72771CGPG2166 SENSE 0.993 0 S 1.381 0.003 S 1.536 0.003 S 432 70217 CGPG6SENSE 0.275 0.067 T 0.126 0.289 / 0.362 0.215 / 433 72711 CGPG1846 SENSE0.774 0.001 S 0.579 0.004 S 0.429 0.038 S 438 72994 CGPG5803 SENSE 0.1160.381 / 0.708 0.068 T 0.744 0.069 T 510 72921 CGPG5781 SENSE 0.265 0.057T 0.31 0.162 T 0.367 0.11 T 414 19951 CGPG3941 SENSE 0.729 0.006 S 0.4730.017 s 0.846 0.006 S 273 70423 CGPG3165 SENSE 0.316 0.012 S 0.521 0.022S 0.89 0.018 S 416 70543 CGPG3815 SENSE 1.584 0 S 0.86 0 S 0.82 0.002 S368 71617 CGPG1227 SENSE 0.204 0.136 T 0.408 0.025 S 0.458 0.057 T 43971928 CGPG1617 SENSE 0.104 0.265 / 0.786 0 S 0.836 0.001 S 382 72466CGPG4767 SENSE 0.497 0.017 S 0.565 0.017 S 0.963 0.002 S 383 72524CGPG4770 SENSE 0.438 0.02 S 0.377 0.025 S 0.385 0.043 S 409 72744CGPG5563 SENSE 0.52 0.058 T 0.859 0.026 S 0.454 0.189 T 444 72967CGPG5742 SENSE 0.955 0 S 0.629 0.009 S 0.403 0.189 T 435 73518 CGPG6497SENSE 0.114 0.278 / 0.319 0.01 S 0.195 0.114 T 306 74136 CGPG6632 SENSE0.606 0.007 S 0.523 0.036 S 0.598 0.025 S 398 74240 CGPG5454 SENSE−0.099 0.644 / 1.277 0.003 S 1.498 0.006 S 431 74369 CGPG6076 SENSE0.623 0.002 S 0.62 0.04 S 0.737 0.096 T S: represents the transgenicplants showed statistically significant trait improvement as compared tothe reference (p < 0.05) T: represents the transgenic plants showed atrend of trait improvement compared to the reference with p < 0.2 /:represents the transgenic plants didn't show any alteration or hadunfavorable change in traits examined as compared to the reference inthe current dataset.

G. Cold Germination Tolerance Screen

This example sets forth a screen to identify Arabidopsis plantstransformed with the genes of interests are resistant to cold stressbased on their rate of development, root growth and chlorophyllaccumulation under low temperature conditions.

T2 seeds were plated and all seedlings used in the experiment were grownat 8° C. Seeds were first surface disinfested using chlorine gas andthen seeded on assay plates containing an aqueous solution of ½×Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo.,USA G/5788), 1% Phytagel™ (Sigma-Aldrich, P-8169), and 10 ug/mlglufosinate with the final pH adjusted to 5.8 using KOH. Test plateswere held vertically for 28 days at a constant temperature of 8° C., aphotoperiod of 16 hr, and average light intensity of approximately 100umol/m²/s. At 28 days post planting, root length was measured, growthstage was observed, the visual color was assessed, and a whole platephotograph was taken.

The root length at day 28 was analyzed as a quantitative responseaccording to example 1M. The growth stage at day 7 was analyzed as aqualitative response according to example 1L. Table 9 provides a list ofrecombinant DNA constructs that improve cold stress tolerance intransgenic plants.

TABLE 9 Growth stage Root Length Pep at day 28 at day 28 SEQ RS p- p- IDConstruct_id Gene Orientation mean value c delta value c 288 19631CGPG3627 SENSE 2.229 0.052 T 0.094 0.252 / 289 70121 CGPG2380 SENSE2.732 0.042 S 0.126 0.238 / 290 70654 CGPG4352 SENSE 2.474 0.026 S 0.2630.019 S 291 70696 CGPG4590 SENSE 3.092 0.01 S 0.086 0.145 T 292 70713CGPG1462 ANTI-SENSE 4 0 S 0.268 0.014 S 293 70740 CGPG3700 SENSE 2.4850.024 S 0.244 0.012 S 294 71321 CGPG4418 SENSE 1.837 0.126 T 0.014 0.474/ 295 71835 CGPG4634 SENSE 3.349 0.002 S 0.353 0.036 S 296 72934CGPG5798 SENSE 2.222 0.023 S 0.243 0.101 T 297 72945 CGPG5787 SENSE3.478 0.001 S 0.236 0.011 S 298 72980 CGPG5773 SENSE 3.265 0.003 S 0.2390.006 S 299 73504 CGPG6480 SENSE 4 0 S 0.521 0 S 300 73507 CGPG6504SENSE 4 0 S 0.404 0.001 S 301 73573 CGPG6462 SENSE 4 0 S 0.268 0.004 S302 73586 CGPG6471 SENSE 4 0 S 0.314 0.064 T 303 73770 CGPG5435 SENSE3.091 0.01 S 0.303 0.085 T 304 74105 CGPG6574 SENSE 3.329 0.002 S 0.1090.116 T 305 74111 CGPG6622 SENSE 2.226 0.021 S 0.445 0.006 S 306 74136CGPG6632 SENSE 3.192 0.005 S 0.328 0.002 S 307 74139 CGPG6561 SENSE 4 0S 0.254 0.092 T 308 74267 CGPG5364 SENSE 3.054 0.002 S 0.3 0 S 309 74291CGPG5363 SENSE 4 0 S 0.142 0.142 T 310 74318 CGPG5826 SENSE 4 0 S 0.2720.008 S 311 74319 CGPG5831 SENSE 3.207 0.005 S 0.201 0.002 S 312 74324CGPG5885 SENSE 3.144 0.007 S 0.232 0.017 S 313 74512 CGPG32 SENSE 4 0 S0.332 0.011 S 314 74583 CGPG6649 SENSE 3.249 0.004 S 0.28 0.001 S 31570427 CGPG3067 SENSE 1.567 0.108 T 0.222 0.044 S 352 73515 CGPG6473SENSE 4 0 S 0.324 0.003 S 353 74684 CGPG6360 SENSE 2.927 0.004 S 0.4260.003 S 373 72633 CGPG4853 SENSE 2.121 0.027 S 0.289 0.048 S 405 71508CGPG1541 SENSE 1.99 0.039 S 0.263 0.033 S 406 74248 CGPG5476 SENSE 2.3850.011 S 0.217 0.017 S 434 70932 CGPG4089 SENSE 3.268 0.003 S 0.146 0.067T 435 73518 CGPG6497 SENSE 3.373 0.002 S 0.352 0.032 S 439 71928CGPG1617 SENSE 3.062 0.011 S 0.18 0.002 S 504 74259 CGPG5343 SENSE 3.5110 S 0.308 0.024 S 505 72463 CGPG4760 SENSE 2.736 0.01 S 0.032 0.386 /506 72902 CGPG5597 SENSE 3.105 0.009 S 0.278 0.037 S 507 74572 CGPG6640SENSE 4 0 S 0.125 0.155 T 508 73055 CGPG5768 SENSE 3.173 0.006 S 0.4070.004 S 413 19707 CGPG4179 SENSE 1.829 0.061 T 0.169 0.018 S 360 19779CGPG4113 SENSE 4 0 S 0.213 0.017 S 361 19833 CGPG4074 SENSE / / / 0.2920.022 S 363 19879 CGPG4009 SENSE 4 0 S 0.34 0.001 S 514 19980 CGPG3914SENSE 0.798 0.122 T 0.278 0.011 S 273 70423 CGPG3165 SENSE 2.906 0.004 S0.105 0.114 T 459 70725 CGPG2097 ANTI-SENSE 1.949 0.044 S 0.122 0.148 T461 71112 CGPG934 SENSE 2.579 0.018 S 0.185 0.17 T 444 72967 CGPG5742SENSE 4 0 S 0.287 0.007 S 438 72994 CGPG5803 SENSE 1.072 0.098 T 0.1610.04 S 521 73488 CGPG6394 SENSE 4 0 S 0.211 0.012 S 477 74117 CGPG6575SENSE 2.567 0.02 S 0.123 0.04 S S: represents the transgenic plantsshowed statistically significant trait improvement as compared to thereference (p < 0.05) T: represents the transgenic plants showed a trendof trait improvement as compared to the reference with p < 0.2 /:represents data points not determined or the transgenic plants didn'tshow any alteration or had unfavorable change in traits examinedcompared to the reference in the current dataset

H. Shade Tolerance Screen

Plants undergo a characteristic morphological response in shade thatincludes the elongation of the petiole, a change in the leaf angle, anda reduction in chlorophyll content. While these changes may confer acompetitive advantage to individuals, in a monoculture the shadeavoidance response is thought to reduce the overall biomass of thepopulation. Thus, genetic alterations that prevent the shade avoidanceresponse are associated with higher yields. Genes that favor growthunder low light conditions may also promote yield, as inadequate lightlevels frequently limit yield. This protocol describes a screen to lookfor Arabidopsis plants that show an attenuated shade avoidance responseand/or grow better than control plants under low light intensity. Ofparticular interest, we were looking for plants that didn't extend theirpetiole length, had an increase in seedling weight relative to thereference and had leaves that were more close to parallel with the platesurface.

T2 seeds were plated on glufosinate selection plates with ½ MS medium.Seeds were sown on ½×MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants weregrown on vertical plates at a temperature of 22° C. at day, 20° C. atnight and under low light (approximately 30 uE/m²/s, far/red ratio(655/665/725/735) ˜0.35 using PLAQ lights with GAM color filter #680).Twenty-three days after seedlings were sown, measurements were recordedincluding seedling status, number of rosette leaves, status of flowerbud, petiole leaf angle, petiole length, and pooled fresh weights. Adigital image of the whole plate was taken on the measurement day.Seedling weight and petiole length were analyzed as quantitativeresponses according to example 1M. The number of rosette leaves,flowering bud formation and leaf angel were analyzed as qualitativeresponses according to example 1L.

Table 10 provides a list of recombinant DNA constructs that improveshade tolerance in plants

TABLE 10 Number of flowerbud formation Leaf Angle Petiole length rosetteleaves seedling weight Pep at day 23 at day 23 at day 23 at day 23 atday 23 SEQ RS p- RS p- RS p- RS p- p- ID Construct_id Orientation meanvalue c mean value c mean value c mean value c delta value c 376 70426SENSE 0.719 0.09 T 0.163 0.244 / −0.206 0.185 T −0.225 0.984 / 0.4840.003 S 377 70772 SENSE −0.501 0.858 / −0.066 0.724 / −0.692 0.004 S−0.626 0.983 / −0.693 0.023 / 378 71137 SENSE 0.26 0.384 / 0.11 0.168 T−0.869 0.029 S 1.064 0.073 T −0.453 0.288 / 379 71529 SENSE 0.89 0.108 T−0.014 1 / −0.44 0.006 S 1.817 0.024 S −0.022 0.914 / 380 71601 SENSE1.79 0.066 T 0.195 0.172 T −0.057 0.59 / −0.483 0.961 / 0.264 0.308 /381 72362 SENSE 1.763 0.072 T 0.218 0.262 / −0.109 0.242 / 0.003 0.484 /0.311 0.119 T 374 72456 SENSE 0.563 0.313 / 0.751 0.186 T −0.848 0.001 S−0.624 0.999 / −0.85 0.025 / 382 72466 SENSE −1.382 1 / −0.092 0.645 /−0.894 0 S 1.212 0.122 T −0.879 0.027 / 383 72524 SENSE −0.153 1 / 0.1520.09 T −0.952 0.002 S −0.57 1 / −1.269 0.01 / 373 72633 SENSE 3.513 0 S−0.431 0.977 / −0.426 0.001 S −0.735 0.994 / 0.269 0.129 T 375 72963SENSE −0.911 0.988 / 0.236 0.396 / −0.753 0.003 S −0.083 0.665 / −0.4850.015 / 384 73085 SENSE −0.073 0.756 / 1.212 0.122 T 0.175 0.097 / 4 0 S0.51 0.027 S 385 74241 SENSE −0.195 0.935 / −0.098 0.64 / −1.077 0.01 S−0.821 0.999 / −1.024 0.002 / 386 74247 SENSE 0.43 0.16 T 0.203 0.095 T−0.22 0.197 T 1.166 0.07 T 0.483 0.018 S 387 74284 SENSE 0.062 0.22 /2.077 0.038 S −0.014 0.943 / 2.348 0.04 S 0.03 0.938 / 388 74652 SENSE0.015 0.442 / 0.093 0.455 / −0.073 0.621 / 0.967 0.184 T 0.173 0.569 /363 19879 SENSE 0.585 / / 0 / / 0.152 / / 0 / / 0.708 / / 293 70740SENSE 0.44 0.137 T 0.609 0.103 T 0.161 0.186 / / / / 0.45 0.001 S 31671811 SENSE −0.025 0.637 / 0.725 0.113 T −0.205 0.01 S / / / −0.5170.037 / 397 71840 SENSE 1.498 0.12 T 0.712 0.178 T −0.379 0.001 S −0.1880.765 / −0.95 0.124 / 468 72450 SENSE 0.068 0.349 / −0.042 1 / 0.1660.016 / 1.62 0.049 S 0.501 0.038 S 370 72757 SENSE 3.595 0 S 0.872 0.079T 0.064 0.138 / −0.767 0.997 / 0.546 0.001 S 444 72967 SENSE 1.829 0.063T 1.123 0.065 T 0.133 0.196 / −0.18 0.794 / 0.509 0.008 S 511 72968SENSE 0.185 0.005 S 0.48 0.179 T 0.22 0.162 / 1.698 0.085 T 0.382 0.031S 300 73507 SENSE 0.248 0.086 T 0.307 0.287 / −0.304 0.031 S / / / 0.050.86 / 307 74139 SENSE −0.011 0.556 / −0.071 1 / −0.124 0.068 T −0.511 1/ −0.413 0.124 / 535 74474 SENSE 0.755 0.154 T −0.145 0.857 / 0.1420.396 / / / / 0.765 0.005 S 400 74610 SENSE 0.572 0.222 / 0.177 0.22 /−0.18 0.071 T / / / −0.341 0.173 / S: represents the transgenic plantsshowed statistically significant trait improvement as compared to thereference (p < 0.05) T: represents the transgenic plants showed a trendof trait improvement as compared to the reference with p < 0.2 /:represents data points not determined or the transgenic plants didn'tshow any alteration or had unfavorable change in traits examinedcompared to the reference in the current dataset.

I. Early Plant Growth and Development Screen

This example sets forth a plate based phenotypic analysis platform forthe rapid detection of phenotypes that are evident during the first twoweeks of growth. In this screen, we were looking for genes that conferadvantages in the processes of germination, seedling vigor, root growthand root morphology under non-stressed growth conditions to plants. Thetransgenic plants with advantages in seedling growth and developmentwere determined by the seedling weight and root length at day 14 afterseed planting.

T2 seeds were plated on glufosinate selection plates and grown understandard conditions (˜100 □E/m²/s, 16 h photoperiod, 22° C. at day, 20°C. at night). Seeds were stratified for 3 days at 4° C. Seedlings weregrown vertically (at a temperature of 22° C. at day 20° C. at night).Observations were taken on day 10 and day 14. Both seedling weight androot length at day 14 were analyzed as quantitative responses accordingto example 1M.

Table 11 provides a list recombinant DNA constructs that improve earlyplant growth and development.

TABLE 11 Pep Root Length Seedling Weight SEQ p- p- ID Construct_id geneOrientation delta value c delta value c 432 70217 CGPG6 SENSE 0.0380.469 / 0.375 0.047 S 433 72711 CGPG1846 SENSE 0.132 0.021 S 0.601 0.001S 434 70932 CGPG4089 SENSE 0.328 0.005 S 0.473 0.017 S 435 73518CGPG6497 SENSE 0.287 0 S 0.634 0.036 S 436 19771 CGPG4011 SENSE 0.2180.076 T 0.581 0.018 S 437 73549 CGPG6460 SENSE 0.139 0.003 S 0.349 0.03S 438 72994 CGPG5803 SENSE 0.44 0.004 S 0.791 0.001 S 439 71928 CGPG1617SENSE 0.073 0.427 / 0.494 0.005 S 440 72903 CGPG5584 SENSE 0.298 0.002 S0.399 0.044 S 441 73017 CGPG5733 SENSE 0.284 0.004 S 0.199 0.488 / 44274587 CGPG6774 SENSE 0.111 0.075 T 0.538 0.006 S 443 72453 CGPG4735SENSE 0.215 0.005 S 0.416 0.069 T 444 72967 CGPG5742 SENSE 0.103 0.212 /0.568 0.008 S 445 72961 CGPG5591 SENSE 0.177 0.046 S 0.548 0.006 S 44673070 CGPG5627 SENSE 0.221 0.007 S 0.652 0.005 S 447 73475 CGPG6385SENSE 0.084 0.014 S 0.336 0.014 S 448 72916 CGPG1814 SENSE 0.182 0.067 T0.353 0.012 S 449 72969 CGPG5789 SENSE 0.133 0.245 / 0.45 0.069 T 45074449 CGPG6659 SENSE 0.314 0.002 S 0.579 0.027 S 451 16615 CGPG2539SENSE 0.223 0.023 S 0.571 0.009 S 452 19187 CGPG3310 SENSE 0.264 0.001 S0.51 0.08 T 453 19648 CGPG3134 SENSE 0.218 0.013 S 0.27 0.106 T 45470354 CGPG3995 SENSE 0.152 0.046 S 0.406 0.029 S 455 70421 CGPG2942SENSE 0.179 0.225 / 0.581 0.013 S 456 70459 CGPG3758 SENSE 0.187 0.036 S−0.034 0.884 / 457 70465 CGPG3775 SENSE −0.009 0.899 / 0.188 0.196 T 45870683 CGPG4587 SENSE 0.133 0.088 T 0.402 0.014 S 459 70725 CGPG2097ANTI-SENSE 0.326 0.001 S 0.116 0.548 / 460 70852 CGPG1465 SENSE 0.237 0S 0.297 0.127 T 461 71112 CGPG934 SENSE 0.199 0.013 S 0.316 0.034 S 46271127 CGPG945 SENSE 0.097 0.02 S 0.4 0.054 T 463 71132 CGPG1561 SENSE0.195 0.02 S 0.08 0.524 / 464 71217 CGPG95 SENSE 0.234 0 S 0.566 0.036 S465 71645 CGPG4688 SENSE 0.475 0.003 S 0.361 0.133 T 466 71726 CGPG3894SENSE 0.223 0.056 T 0.458 0.033 S 467 72432 CGPG4562 SENSE 0.209 0 S0.581 0.041 S 468 72450 CGPG4732 SENSE 0.335 0 S 0.79 0 S 469 72455CGPG4742 SENSE 0.278 0.019 S 0.482 0.051 T 470 72727 CGPG5522 SENSE0.123 0.002 S 0.315 0.004 S 471 72817 CGPG4987 SENSE 0.254 0.023 S 0.4850 S 472 72992 CGPG5777 SENSE 0.219 0.023 S 0.664 0.015 S 473 73007CGPG5760 SENSE 0.139 0.093 T 0.462 0.008 S 474 73073 CGPG5688 SENSE0.164 0.022 S 0.285 0.247 / 475 73506 CGPG6496 SENSE 0.512 0 S 0.986 0 S476 74107 CGPG6590 SENSE 0.282 0.002 S 0.538 0.057 T 477 74117 CGPG6575SENSE 0.211 0.002 S 0.449 0.005 S 478 74131 CGPG6592 SENSE 0.142 0.047 S0.586 0.003 S 479 74344 CGPG5929 SENSE 0.27 0.01 S 0.474 0.105 T 32313502 CGPG1354 SENSE 0.57 0.002 S 0.625 0 S 330 18259 CGPG3368 SENSE0.226 0.026 S 0.551 0.021 S 361 19833 CGPG4074 SENSE 0.259 0 S 0.4720.016 S 334 70417 CGPG3427 SENSE 0.187 0.056 T 0.113 0.65 / 273 70423CGPG3165 SENSE 0.03 0.747 / 0.185 0.007 S 515 70435 CGPG3701 SENSE 0.1310.051 T 0.394 0.014 S 493 70601 CGPG2917 SENSE 0.177 0 S 0.365 0.063 T291 70696 CGPG4590 SENSE 0.079 0.205 / 0.331 0.024 S 365 70738 CGPG3195SENSE 0.012 0.864 / 0.472 0.003 S 293 70740 CGPG3700 SENSE 0.103 0.082 T0.387 0.021 S 419 71134 CGPG817 SENSE 0.063 0.548 / 0.279 0.066 T 42272086 CGPG5236 SENSE 0.083 0.234 / 0.398 0.05 S 505 72463 CGPG4760 SENSE−0.131 0.416 / 0.422 0.003 S 285 73014 CGPG5692 SENSE 0.11 0.391 / 0.3730.096 T 521 73488 CGPG6394 SENSE 0.018 0.793 / 0.398 0.1 T 299 73504CGPG6480 SENSE −0.152 0.479 / 0.538 0.012 S 300 73507 CGPG6504 SENSE0.15 0.002 S 0.053 0.854 / 301 73573 CGPG6462 SENSE 0.112 0.07 T 0.3750.006 S 302 73586 CGPG6471 SENSE 0.309 0 S 0.611 0 S 303 73770 CGPG5435SENSE 0.21 0.059 T 0.281 0.484 / 509 74103 CGPG6558 SENSE 0.374 0 S0.561 0.024 S 428 74140 CGPG6569 SENSE 0.16 0.017 S 0.376 0.066 T 52774262 CGPG5353 SENSE 0.203 0.012 S 0.375 0.045 S 430 74265 CGPG5356SENSE 0.107 0.259 T 0.432 0.067 T 529 74302 CGPG5384 SENSE 0.115 0.101 T0.269 0.056 T 431 74369 CGPG6076 SENSE 0.138 0.03 S 0.195 0.21 / 53474465 CGPG6692 SENSE 0.2 0.02 S 0.688 0.042 S 507 74572 CGPG6640 SENSE0.162 0.023 S 0.617 0 S 314 74583 CGPG6649 SENSE 0.144 0.023 S 0.4030.008 S S: represents the transgenic plants showed statisticallysignificant trait improvement as compared to the reference (p < 0.05) T:represents the transgenic plants showed a trend of trait improvement ascompared to the reference with p < 0.2 /: represents the transgenicplants didn't show any alteration or had unfavorable change in traitsexamined as compared to the reference in the current dataset

J. Late Plant Growth and Development Screen

This example sets forth a soil based phenotypic platform to identifygenes that confer advantages in the processes of leaf development,flowering production and seed maturity to plants. Arabidopsis plantswere grown on a commercial potting mixture (Metro Mix 360, Scotts Co.,Marysville, Ohio) consisting of 30-40% medium grade horticulturalvermiculite, 35-55% sphagnum peat moss, 10-20% processed bark ash, 1-15%pine bark and a starter nutrient charge. Soil was supplemented withOsmocote time-release fertilizer at a rate of 30 mg/ft³. T2 seeds wereimbibed in 1% agarose solution for 3 days at 4° C. and then sown at adensity of ˜5 per 2½″ pot. Thirty-two pots were ordered in a 4 by 8 gridin standard greenhouse flat. Plants were grown in environmentallycontrolled rooms under a 16 h day length with an average light intensityof ˜200 μmoles/m²/s. Day and night temperature set points were 22° C.and 20° C., respectively. Humidity was maintained at 65%. Plants werewatered by sub-irrigation every two days on average until mid-flowering,at which point the plants were watered daily until flowering wascomplete.

Application of the herbicide glufosinate was performed to select T2individuals containing the target transgene. A single application ofglufosinate was applied when the first true leaves were visible. Eachpot was thinned to leave a single glufosinate-resistant seedling ˜3 daysafter the selection was applied.

The rosette radius was measured at day 25. The silique length wasmeasured at day 40. The plant parts were harvested at day 49 for dryweight measurements if flowering production was stopped. Otherwise, thedry weights of rosette and silique were carried out at day 53. The seedswere harvested at day 58. All measurements were analyzed as quantitativeresponses according to example 1M.

Table 12 provides a list of recombinant DNA constructs that improve lateplant growth and development.

TABLE 12 Rosette Dry Rosette Seed Dry Silique Dry Slilique Pep WeightRadius Weight Weight Length SEQ p- p- p- p- p- ID Construct_idOrientation delta value c delta value c delta value c delta value cdelta value c 480 14320 SENSE −0.145 0.94 / 0.137 0.038 S −0.702 1 /0.477 0.002 S 0.016 0.276 / 481 16756 SENSE 0.485 0.016 S 0.223 0.025 S0.148 0.042 S 0.481 0.002 S 0.147 0.013 S 482 17448 SENSE −0.288 0.991 /−0.054 0.829 / 0.376 0.008 S 0.185 0.034 S −0.064 0.981 / 483 17633SENSE 0.018 0.359 / 0.13 0.106 T 0.416 0.055 T 0.399 0.044 S 0.116 0.08T 484 18876 SENSE 0.257 0.016 S 0.026 0.448 / −0.213 0.786 / 0.386 0 S−0.015 0.705 / 485 19120 ANTI-SENSE −0.252 0.936 / 0.022 0.006 S −1.0420.95 / 0.165 0.076 T 0.046 0.021 S 486 19221 SENSE −0.316 0.986 / 0.1530.097 T −0.35 0.903 / 0.394 0.068 T 0.183 0.028 S 487 70206 SENSE 0.1250 S 0.074 0.18 T 0.712 0.022 S 0.12 0.026 S 0.019 0.403 / 488 70223SENSE 0.197 0.026 S 0.23 0.018 S −0.781 0.998 / 0.134 0.039 S −0.2050.915 / 489 70347 SENSE 0.156 0.038 S −0.082 0.868 / −0.153 0.752 /0.405 0.006 S 0.056 0.068 T 490 70406 SENSE −0.275 0.948 / −0.245 0.992/ 0.759 0.025 S −0.282 0.949 / −0.121 0.939 / 491 70469 SENSE 0.0320.392 / 0.348 0.004 S −0.733 0.996 / 0.325 0.059 T −0.141 0.922 / 49270564 SENSE 0.17 0.037 S 0.051 0.234 / 0.772 0 S −0.381 0.977 / −0.0150.655 / 493 70601 SENSE 0.231 0.086 T 0.247 0.004 S −0.257 0.959 / 0.3230.024 S 0.082 0.03 S 494 70612 SENSE 0.053 0.112 T 0.082 0.096 T 1.0490.011 S −0.212 0.992 / 0.07 0.004 S 495 70720 ANTI-SENSE −0.16 0.898 /0.028 0.384 / 1.312 0.009 S 0.219 0.121 T 0.128 0.018 S 496 70735 SENSE−0.058 0.672 / 0.156 0.087 T 0.421 0.036 S 0.532 0.003 S 0.033 0.038 S497 70846 SENSE 0.086 0.255 / 0.134 0.063 T −0.33 0.918 / 0.142 0.151 T0.011 0.439 / 498 70923 SENSE 0.484 0.011 S 0.108 0.081 T −0.4 0.844 /0.091 0.198 T 0.099 0.001 S 499 71149 SENSE −1.085 0.993 / −0.043 0.681/ 0.346 0.017 S 0.136 0.133 T 0.066 0.077 T 500 71608 SENSE −0.849 0.907/ −0.132 0.976 / 0.816 0.006 S 0.279 0.004 S 0.038 0.238 / 501 71739SENSE −0.275 0.937 / −0.107 0.955 / 0.334 0 S −0.075 0.815 / −0.1190.874 / 502 72014 SENSE −0.038 0.94 / 0.07 0.278 / 0.732 0.06 T −0.0260.584 / −0.013 0.596 / 503 72051 SENSE 0.026 0.28 / 0.311 0.003 S 0.2220.236 / 0.453 0.009 S 0.052 0.168 T 432 70217 SENSE −0.202 0.893 / 0.2030.024 S −0.079 0.743 / 0.27 0.07 T −0.063 0.856 / 454 70354 SENSE 0.1340.147 T −0.119 0.684 / 0.48 0.02 S −0.119 0.736 / 0.014 0.3 / 334 70417SENSE −0.35 0.988 / −0.041 0.637 / 0.69 0.012 S −0.136 0.978 / 0.0040.411 / 515 70435 SENSE 0.664 0.014 S 0.146 0.036 S 0.138 0.097 T 0.330.038 S 0.039 0.16 T 457 70465 SENSE −0.106 0.79 / / / / 0.883 0.001 S−0.27 0.945 / −0.097 0.818 / 460 70852 SENSE 0.178 0.031 S 0.145 0.034 S−0.525 0.904 / −0.315 0.861 / −0.059 0.774 / 405 71508 SENSE 0.195 0.162T 0.251 0.017 S −0.515 0.929 / −0.322 0.997 / −0.196 0.982 / 295 71835SENSE 0.139 0.055 T 0.163 0.049 S 0.538 0.019 S 0.27 0.068 T 0 0.496 /467 72432 SENSE 0.146 0.021 S 0.139 0.02 S 0.325 0.012 S 0.074 0.149 T−0.106 0.879 / 443 72453 SENSE 0.204 0.037 S 0.116 0.046 S −2.198 0.995/ 0.016 0.448 / −0.013 0.534 / 433 72711 SENSE 0.292 0.058 T 0.143 0.024S −0.114 0.76 / −0.04 0.667 / −0.093 0.946 / 449 72969 SENSE 0.046 0.072T −0.158 0.887 / 0.39 0.031 S 0.477 0.001 S 0.095 0.054 T 426 72987SENSE 0.385 0.006 S / / / 0.098 0.104 T 0.153 0.016 S 0.057 0.061 T 52574022 SENSE 0.11 0.226 / −0.069 0.844 / 0.71 0.009 S −0.05 0.613 /−0.004 0.544 / 287 74251 SENSE −0.56 0.961 / −0.184 0.916 / 0.611 0.001S 0.174 0.229 / −0.137 0.923 / 537 74507 SENSE 0.255 0.017 S 0.178 0.03S 0.318 0.032 S 0.188 0.011 S 0.013 0.078 T 313 74512 SENSE −0.247 0.953/ 0.107 0.015 S −0.073 0.86 / 0.113 0.034 S 0.05 0.175 T S: representsthe transgenic plants showed statistically significant trait improvementas compared to the reference (p < 0.05) T: represents data points notdetermined or the transgenic plants showed a trend of trait improvementcompared to the reference with p < 0.2 /: represents the transgenicplants didn't show any alteration or had unfavorable change in traitsexamined as compared to the reference in the current dataset

K. Limited Nitrogen Tolerance Screen

Under low nitrogen conditions, Arabidopsis seedlings become chloroticand have less biomass. This example sets forth the limited nitrogentolerance screen to identify Arabidopsis plants transformed with thegene of interest that are altered in their ability to accumulate biomassand/or retain chlorophyll under low nitrogen condition.

T2 seeds were plated on glufosinate selection plates containing0.5×N-Free Hoagland's T 0.1 mM NH₄NO₃ T 0.1% sucrose T 1% phytagel mediaand grown under standard light and temperature conditions. At 12 days ofgrowth, plants were scored for seedling status (i.e., viable ornon-viable) and root length. After 21 days of growth, plants were scoredfor visual color, seedling weight, number of green leaves, number ofrosette leaves, root length and formation of flowering buds. Aphotograph of each plant was also taken at this time point.

The seedling weight and root length were analyzed as quantitativeresponses according to example 1M. The number green leaves, the numberof rosette leaves and the flowerbud formation were analyzed asqualitative responses according to example 1L.

Table 13 provides a list of recombinant DNA constructs that improve lownitrogen availability tolerance in plants.

TABLE 13 Number of Flowerbud Number of rosette Pep formation greenleaves Root Length leaves Seedling Weight SEQ RS p- RS p- p- RS p- p- IDConstruct_id Orientation mean value c mean value c delta value c meanvalue c delta value c 375 72963 SENSE 1.1 0.004 S 0.293 0.021 S −0.4460.002 S −0.246 0.786 / 0.137 0.001 S 389 70437 SENSE −0.28 0.982 / −0.080.769 / 0.259 0.006 S 0.5 0.005 S 0.133 0.003 S 390 71633 SENSE 0.260.26 / 0.254 0.114 T −0.1 0.539 / 0.647 0.06 T 0.106 0.023 S 391 72948SENSE 0.587 0.06 T 0.539 0.029 S −0.237 0.003 S 0.479 0.1 T 0.078 0.121T 392 72519 SENSE 0.749 0.033 S 0.209 0.104 T −0.09 0.274 / 0.276 0.264/ 0.116 0.006 S 393 10475 SENSE 1.256 0.026 S 0.588 0.005 S −0.378 0.002S 0.081 0.319 / 0.018 0.75 / 394 11120 ANTI-SENSE 0.795 0.033 S 0.6080.015 S −0.45 0.001 S 0.287 0.106 T −0.041 0.387 / 395 19736 SENSE −0.240.907 / 0.355 0.033 S 0.014 0.864 / 0.64 0.006 S −0.075 0.005 / 39671606 SENSE 0.605 0.088 T 0.176 0.11 T −0.033 0.708 / 1.239 0.005 S0.133 0.002 S 397 71840 SENSE 0.408 0.235 / 0.879 0.006 S −0.137 0.248 /0.524 0.032 S 0.066 0.198 T 398 74240 SENSE −0.06 0.602 / 0.13 0.076 T−0.107 0.306 / 0.714 0.034 S 0.108 0.002 S 399 74331 SENSE −0.44 1 /0.054 0.203 / 0.132 0.055 T 1.045 0.021 S 0.134 0.003 S 400 74610 SENSE−0.59 1 / −0.08 0.922 / 0.289 0 S 1.241 0.017 S 0.137 0.001 S 401 75527SENSE 0.242 0.228 / 0.376 0.028 S −0.183 0.045 / 0.352 0.083 T −0.0050.8 / 316 71811 SENSE −0.45 0.91 / / / / −0.112 0.202 / 0.438 0.014 S−0.054 0.161 / 505 72463 SENSE −0.16 0.976 / / / / 0.112 0.109 T 0.3660.024 S 0.13 0.006 S 351 75701 SENSE 0.736 0.048 S 0.07 0.861 / −0.40.018 S / / / −0.109 0.193 / S: represents the transgenic plants showedstatistically significant trait improvement as compared to the reference(p < 0.05) T: represents the transgenic plants showed a trend of traitimprovement compared than the reference with p < 0.2 /: represents datapoints not determined or the transgenic plants didn't show anyalteration or had unfavorable change in traits examined as compared tothe reference in the current dataset

L. Statistic Analysis for Qualitative Responses

Table 14 provides a list of responses that were analyzed as qualitativeresponses

TABLE 14 response Screen categories (success vs. failure) wiltingresponse Risk Soil drought tolerance screen non-wilted vs. wilted Scoregrowth stage at day 14 heat stress tolerance screen 50% of plants reachstage1.03 vs. not growth stage at day 14 salt stress tolerance screen50% of plants reach stage1.03 vs. not growth stage at day 14 PEG inducedosmotic stress tolerance 50% of plants reach stage1.03 vs. not screengrowth stage at day 7 cold germination tolerance screen 50% of plantsreach stage 0.5 vs. not number of rosette leaves Shade tolerance screen5 leaves appeared vs. not at day 23 flower bud formation at Shadetolerance screen flower buds appear vs. not day 23 leaf angle at day 23Shade tolerance screen >60 degree vs. <60 degree number of green leavesat limited nitrogen tolerance screen 6 or 7 leaves appeared vs. not day21 number of rosette leaves limited nitrogen tolerance screen 6 or 7leaves appeared vs. not at day 21 Flower bud formation at limitednitrogen tolerance screen flower buds appear vs. not day 21

Plants were grouped into transgenic and reference groups and were scoredas success or failure according to criteria in Table 14. First, the risk(R) was calculated, which is the proportion of plants that were scoredas of failure plants within the group. Then the relative risk (RR) wascalculated as the ratio of R (transgenic) to R (reference). Risk score(RS) was calculated as −log₂ ^(RR). Subsequently the risk scores frommultiple events for each transgene of interest were evaluated forstatistical significance by t-test using S-PLUS statistical software(S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). RSwith a value greater than 0 indicates that the transgenic plants performbetter than the reference. RS with a value less than 0 indicates thatthe transgenic plants perform worse than the reference. The RS with avalue equal to 0 indicates that the performance of the transgenic plantsand the reference don't show any difference.

M. Statistic Analysis for Quantitative Responses

Table 15 provides a list of responses that were analyzed as quantitativeresponses.

TABLE 15 response screen seed yield Soil drought stress tolerance screenseedling weight at day 14 heat stress tolerance screen root length atday 14 heat stress tolerance screen seedling weight at day 14 saltstress tolerance screen root length at day 14 salt stress tolerancescreen root length at day 11 salt stress tolerance screen seedlingweight at day 14 PEG induced osmotic stress tolerance screen root lengthat day 11 PEG induced osmotic stress tolerance screen root length at day14 PEG induced osmotic stress tolerance screen rosette area at day 8cold shock tolerance screen rosette area at day28 cold shock tolerancescreen difference in rosette area cold shock tolerance screen from day 8to day 28 root length at day 28 cold germination tolerance screenseedling weight at day 23 Shade tolerance screen petiole length at day23 Shade tolerance screen root length at day 14 Early plant growth anddevelopment screen Seedling weight at day14 Early plant growth anddevelopment screen Rosette dry weight at Late plant growth anddevelopment screen day 53 rosette radius at day 25 Late plant growth anddevelopment screen seed dry weight at day 58 Late plant growth anddevelopment screen silique dry weight at day 53 Late plant growth anddevelopment screen silique length at day 40 Late plant growth anddevelopment screen Seedling weight at day 21 Limited nitrogen tolerancescreen Root length at day 21 Limited nitrogen tolerance screen

The measurements (M) of each plant were transformed by log₂ calculation.The Delta was calculated as log 2M(transgenic)-log 2M(reference).Subsequently the mean delta from multiple events of the transgene ofinterest was evaluated for statistical significance by t-test usingS-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful,Seattle, Wash., USA). The Delta with a value greater than 0 indicatesthat the transgenic plants perform better than the reference. The Deltawith a value less than 0 indicates that the transgenic plants performworse than the reference. The Delta with a value equal to 0 indicatesthat the performance of the transgenic plants and the reference don'tshow any difference.

Example 2 Identification of Homologs

A BLAST searchable “All Protein Database” was constructed of knownprotein sequences using a proprietary sequence database and the NationalCenter for Biotechnology Information (NCBI) non-redundant amino aciddatabase (nr.aa). For each organism from which a DNA sequence providedherein was obtained, an “Organism Protein Database” was constructed ofknown protein sequences of the organism; the Organism Protein Databaseis a subset of the All Protein Database based on the NCBI taxonomy IDfor the organism.

The All Protein Database was queried using amino acid sequence ofcognate protein for gene DNA used in trait-improving recombinant DNA,i.e., sequences of SEQ ID NO: 240 through SEQ ID NO: 478 using “blastp”with E-value cutoff of 1e-8. Up to 1000 top hits were kept, andseparated by organism names. For each organism other than that of thequery sequence, a list was kept for hits from the query organism itselfwith a more significant E-value than the best hit of the organism. Thelist contains likely duplicated genes, and is referred to as the CoreList. Another list was kept for all the hits from each organism, sortedby E-value, and referred to as the Hit List.

The Organism Protein Database was queried using amino acid sequences ofSEQ ID NO: 270 through SEQ ID NO: 538 using “blastp” with E-value cutoffof 1e-4. Up to 1000 top hits were kept. A BLAST searchable database wasconstructed based on these hits, and is referred to as “SubDB”. SubDBwas queried with each sequence in the Hit List using “blastp” withE-value cutoff of 1e-8. The hit with the best E-value was compared withthe Core List from the corresponding organism. The hit is deemed alikely ortholog if it belongs to the Core List, otherwise it is deemednot a likely ortholog and there is no further search of sequences in theHit List for the same organism. Likely orthologs from a large number ofdistinct organisms were identified and are reported by amino acidsequences of SEQ ID NO: 539 to SEQ ID NO: 22568. The relationship of thehomologs to the identified trait-improving genes on an amino acidsequence basis is found in Table 16 where the amino acid sequence of aprotein encoded by a trait-improving DNA, e.g., SEQ ID NO:270, isfollowed by the amino acid sequences of protein encoded by homologousgenes, e.g., SEQ ID NO:19844, 4248, 2761, 15944, etc. The sourceorganism of each homolog is reported in the Sequence Listing.

TABLE 16 Sequence IDs for homolog proteins Seq ID NO: homolog Seq ID NOs270: 19844 4248 2761 15944 11776 16144 10470 9742 6776 1010 2285 163339154 20620 16454 20025 8388 10646 1208 6001 1706 2448 14768 10226 1262619846 9302 17295 17794 6354 5098 1789 6430 17749 821 10109 7542 1785515562 17462 271: 1715 5418 7208 19338 7440 711 8113 17151 17592 188794807 8671 9936 11315 10681 3177 10519 6830 13563 12162 15155 1860 1807220945 6715 15032 5192 10928 272: 0 273: 13771 8553 4219 10043 10800 834517501 13569 954 17197 6188 3760 13267 16169 8132 2667 11216 15637 46522270 10309 5708 18374 9446 12844 7790 7569 4786 9725 14187 12859 1694818626 13741 5525 7877 4550 15544 9706 7616 14358 15163 13182 14560 167221129 1472 4261 10693 20144 6437 21413 17893 17984 17116 9925 19953 20648983 2837 5663 2943 10465 1841 12497 6435 14763 13495 12676 7513 836316389 8162 7945 14956 15029 12433 22241 16071 13003 16940 18847 123547732 14013 5735 11505 8833 17658 16048 17609 575 6641 6331 3738 1084218927 21518 20097 14117 5309 13744 15880 19484 9648 22509 1221 155156785 852 6466 17423 14164 658 8704 16710 19375 3306 14050 16883 163221722 15481 13636 10680 6347 3552 8885 21794 17703 22557 14777 2118913711 3601 3968 3692 4003 20044 12943 19749 1865 6355 14902 6137 2237019468 10410 21460 10451 17175 20965 12916 1206 6796 11329 9139 1100810569 9058 7988 19743 20088 14111 8231 4522 18497 11952 2866 15466 36092403 16796 13539 14806 5364 12620 20699 12940 15426 4409 12452 5296 9156629 12665 20947 3649 7530 274: 16167 5178 2412 10455 20036 11246 196666400 5573 22539 8547 21845 2413 20290 4036 19351 5886 6071 17184 9738275: 14665 16694 12678 14928 21489 7918 1571 3959 2490 2517 14615 378810022 16096 21248 13293 8541 13446 6120 4360 3812 15574 18938 19203 22842215 10054 14052 9653 10183 17752 20776 4240 22343 8270 9192 17217 737412141 20657 7674 6445 2522 276: 12496 3460 13599 7043 9150 1664 277:16949 9640 8150 2014 12188 5779 17876 14612 18293 11053 15958 1526318370 20984 13094 18734 7380 10318 21641 12737 13028 20561 7087 106869894 7528 12573 16043 14846 20513 2802 8897 14716 10257 16407 2727 61511484 6831 16916 10146 17756 13193 7670 15946 7750 9397 20046 12547 539918644 11883 12531 12530 17188 2130 3805 17493 16821 10181 3639 3934 1419780 278: 16531 9228 5799 19821 10980 17656 3449 19982 13335 20959 1123813084 10281 17610 19623 17614 9736 21375 19978 5859 4943 12390 188061349 8759 21741 20400 13707 9170 15899 11361 4333 10631 12909 2136 1277620323 7676 5847 9065 19902 4545 6768 546 9246 14134 4442 20731 9931 67008677 19305 4828 3655 17550 7579 21629 9044 19475 637 4209 3519 387315884 12247 10177 1407 10312 15957 16955 17469 20241 7267 21862 1686422192 19599 6365 12324 10985 6424 6449 14564 3115 14885 10603 911 186093359 7059 2851 5801 9613 8391 18695 7987 14964 1081 15171 1312 147473060 13390 22115 5060 16536 19729 13468 11109 21989 2230 9462 9096 1877510721 11999 8340 16607 22199 8687 3652 8147 22073 4090 3491 20506 783510890 6781 7839 14478 16371 579 6442 9721 5423 2566 7876 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21495 14630 21041 5676 8447 16750 10436 4523 2686 8237 2948 1355919008 3686 18465 3895 7416 10170 18867 14574 1159 280: 16310 3796 1020519486 13581 11022 21960 15598 12604 9662 20210 21764 8626 17323 1166621316 6121 7438 982 2135 17499 12340 20711 6553 10296 17874 914 177163723 281: 20551 21096 13717 4006 19762 10017 14425 17785 6291 3855 1523211917 21856 18564 19010 3910 13621 19087 670 10539 851 5698 9112 1269414218 11947 18104 21227 22348 14178 1862 9829 9325 14533 13149 867917127 6507 10375 2782 3357 594 7548 9316 5728 7981 8022 13688 1294 1381721893 10781 5263 11292 5492 9542 8000 16102 6328 11687 13695 15807 40683478 6486 13660 22165 17881 19166 3613 7013 6393 15983 17688 5124 424319684 17008 12366 7161 20062 12194 15870 14385 9124 9865 6211 628 6448700 8869 17941 10697 21134 5586 4469 21167 855 20538 9251 1036 106781977 17337 4575 19974 3520 10195 14572 3870 21293 19011 12921 2012011647 15054 13976 21163 20362 12988 15636 2345 4740 3205 17504 195315208 16834 7654 15907 8961 282: 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17456 3953 3584 4314 6428 14221 4758 5800 2236517678 16252 14893 17154 19437 16453 21224 9572 16225 9367 1899 8751 842612483 4441 20592 6867 2168 16928 13225 22460 20208 12970 2591 5304 1349821581 4699 21894 1461 5860 21622 18703 2500 7014 15116 596 14962 551713693 21739 19681 11533 3338 3956 11364 17988 531: 15910 22524 135891006 11575 20349 18523 10911 13067 10217 9609 5233 4966 9015 21390 184619866 21665 7323 5049 9997 5240 6367 19001 2814 21443 1880 1396 361513867 7787 22538 13411 13344 1205 18398 13083 17485 1550 757 11281 1102921967 827 1873 21174 14091 5281 885 7332 8229 21103 4400 1012 15883 78127121 16424 1339 1866 3474 11617 19918 14296 1352 17699 12367 3388 2089620167 6697 19989 9358 15375 4618 11468 13543 3097 15540 19380 14287 505111086 12824 13271 15196 6303 4194 1182 15803 1842 19299 2857 17664 2158911961 19154 12506 21814 6334 21437 20936 11195 20933 21506 22069 54763533 19583 7949 4677 18511 4730 696 9569 17797 18687 7097 20259 1525213221 13305 450 22502 14915 21157 6730 2195 19855 10324 11089 1744 1991716130 21296 2832 830 14553 6434 18295 17956 829 476 12043 2457 1395912384 5220 3209 19341 5731 10908 4395 21843 14568 17630 2371 4694 1239615369 13051 16597 3697 10741 16309 4927 13396 21621 8841 16662 1112014081 19449 21259 15204 6565 18317 14304 4952 7813 7306 12296 12021 664521386 16505 9286 22489 3622 14762 18330 10802 10479 16663 3678 167137751 13703 3630 4691 9472 10709 8542 7060 6112 22457 21974 20476 73336482 14526 7151 2644 835 10655 12264 9315 2786 16253 9488 5634 173723836 7637 4946 18143 11559 18362 12059 10889 5190 13207 16465 17346 33235354 5825 2210 19147 9333 13177 20541 3458 1240 15787 20099 9282 114804994 14011 1145 19545 802 17144 13420 13439 14301 1357 10965 15558 2225122565 810 7570 2692 15666 13168 10912 20574 4894 2526 18982 4020 197962984 11522 11245 21570 8695 16749 17392 2132 20312 4655 16568 2109313882 5730 7976 7079 13035 2800 16658 18348 16408 4157 8112 5264 30512545 2037 11704 9571 21379 3712 7685 4483 18456 22341 3397 11197 13107675 16006 20604 18536 532: 15542 3190 1006 429 7236 21108 10856 102171694 1170 507 478 7946 4963 10952 8465 18461 9739 1850 5240 2382 636719001 19407 14319 21808 15520 1498 14212 14282 19450 526 3987 1803412939 14517 22533 14740 7812 18725 15422 12515 6665 8294 20640 9749 793318742 10601 1528 14184 8297 11430 21253 7897 4901 7308 16574 19527 98828330 6186 16730 3076 15579 16073 19400 17335 2857 17664 21589 1196119154 6997 3197 21437 4440 11195 20933 21506 3533 4677 18511 4730 6969569 17797 18687 7097 20259 2920 15252 13221 3452 14930 450 11335 2077712713 22502 14915 21157 14054 10864 5059 21184 8816 9146 14653 1457920371 3563 12591 16308 7626 4212 19798 19458 5691 18292 7706 5100 454016130 21296 2832 830 14553 6434 18295 17956 829 476 12043 13241 110234301 9298 2269 1306 13291 17064 20779 5613 1281 17745 11486 9723 165867955 12705 15449 988 20859 1768 11129 6547 22518 5552 9854 11376 66695359 9132 10964 1879 22087 13137 17298 3333 13752 20301 19370 2425 204616946 19538 14273 15340 6427 17165 15603 12419 10809 8003 13658 1406414568 17630 2371 4694 12396 15369 16597 3697 10741 12482 16309 492713396 21621 8841 16662 11120 14081 15751 4903 21508 18755 14599 63848692 9484 15204 6565 14320 2978 9294 7482 9493 1277 4952 7813 7306 1229612021 6645 9286 22489 13745 16653 19069 7780 15219 16969 14762 1833010802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 70606112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264 93152786 16253 9488 5634 17372 1733 20453 21787 16624 7001 20828 13302 103574549 788 10490 19133 12318 5515 9280 22095 18903 3706 15256 18593 576411115 12583 11568 13613 2331 5136 8073 15998 5630 11304 19137 5817 55803044 18341 8588 5857 12540 2454 4970 16919 17445 2401 11869 6193 2151610889 5190 13207 16465 9673 11625 9771 2283 11716 19554 905 7481 1240119686 13656 22135 9553 18575 9584 12794 5644 21399 20485 9218 18691 335018263 4846 544 19667 9933 4140 1318 20418 11128 20105 16734 2376 156997061 4232 15357 14036 5339 7107 19030 7165 21370 12103 4848 13211 2253015360 12863 9975 6398 14067 16683 21170 1924 890 8155 15866 10131 71874332 1235 20330 12927 7088 5099 2302 12424 8303 17466 14322 11383 228215956 3414 12982 18548 15665 10961 21084 10824 18440 16819 3730 1394018821 14864 1818 19607 7969 6546 16771 5441 8459 18266 5000 15749 1301414274 8444 4707 13097 15930 11872 2621 7158 6942 18502 5408 10837 2192813800 5188 19614 16117 4719 1833 13499 3107 21492 12503 21929 7921 142914398 22189 6439 12993 12307 19714 6650 18994 747 5932 18630 6683 192115651 5594 6958 13597 19763 10097 14124 14687 1094 5780 7770 7688 151105797 7907 21169 3329 12627 4065 19882 22067 19211 2061 7038 8909 1691413129 19881 11720 16317 1551 13235 14888 1201 20264 7800 7526 2209 588715285 10798 15317 10223 11351 10105 10661 15014 2908 16412 22277 1985112003 19616 11003 7768 6166 21613 4620 13850 16231 7016 2717 20541 34581240 15787 20099 9282 11480 4994 937 16775 18459 14476 9221 17450 628118662 7992 9661 19875 11156 3619 14086 20948 16946 3456 6143 786 67095954 17926 20839 22351 13277 11255 5140 4283 9455 4818 14459 5187 1666810965 15558 17215 14289 11685 4388 14119 18982 2984 11245 304 477 42718112 965 2900 2132 20312 4655 16568 21093 13882 5730 7976 7079 130355498 2800 19337 9600 307 12545 2037 11704 9571 21379 3712 7685 448318456 22341 11197 13107 16006 7623 533: 6090 1006 429 10217 10250 1991018933 314 18461 5240 6367 19001 16913 19119 526 3693 7812 13179 534 300531 2857 17664 21589 11961 19154 21437 11195 20933 21506 3533 4677 185114730 696 9569 17797 18687 7097 20259 15252 13221 450 11335 20777 1271322502 14915 21157 20371 3563 12591 16308 7626 4212 19798 19458 569118292 16130 21296 2832 830 14553 6434 18295 17956 829 476 12043 207791281 17745 16586 7955 12705 15449 988 20859 1768 11129 6547 22518 55529854 11376 6669 5359 9132 10964 1879 22087 13137 17298 3333 13752 2030119370 2425 6946 19538 14273 15603 15340 6427 17165 12419 10809 800313658 14064 4221 7387 13284 20981 2316 3116 8418 3386 6508 18217 357822249 992 13442 3471 14855 19949 5277 19885 2816 10917 9436 18538 1865912816 12308 15943 2515 20364 14701 8193 17579 22076 8890 2649 9329 210738376 15177 10882 859 5990 9814 16053 5209 3144 13848 22428 19076 545417056 14918 4088 11270 5837 9604 22455 19840 12234 1149 801 953 996021301 1656 3755 12278 22119 21098 1005 10264 13037 11702 20954 5581 50631118 6454 20632 1696 19474 14911 9381 4111 13374 2932 3071 18551 223621431 12923 6509 19229 14012 5372 12362 17380 20272 16391 13395 5132 9019540 19228 11589 13306 3179 14568 17630 2371 4694 12396 15369 1178416597 3697 10741 16309 4927 13396 21621 8841 16662 11120 14081 8692 948415204 6565 14320 2978 9294 7482 9493 1277 4952 7813 7306 12296 120216645 9286 22489 13745 16653 19069 7780 15219 16969 14762 18330 1028010802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 70606112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264 93152786 16253 9488 5634 17372 788 9280 22095 18903 3706 15256 18593 576411115 12583 11568 13613 2331 5136 8073 15998 5630 11304 19137 5817 558018341 8588 12540 2454 4970 17445 2401 11869 6193 21516 2447 10889 519013207 16465 9673 9771 11716 18575 9584 12794 21399 20485 9218 18691 335018263 4846 544 19667 9933 4140 1318 20418 11128 20105 16734 2376 156997061 4232 15357 14036 5339 7107 19030 7165 21370 12103 4848 13211 2253015360 12863 9975 6398 14067 16683 21170 1924 890 8155 15866 10131 71874332 1235 20330 12927 7088 5099 2302 12424 8303 17466 14322 11383 228215956 3414 12982 18548 15665 10961 21084 10824 18440 16819 3730 1394018821 14864 1818 19607 7969 6546 16771 5441 8459 18266 5000 15749 1301414274 8444 4707 13097 15930 11872 2621 7158 6942 18502 5408 10837 2192813800 5188 19614 16117 4719 1833 13499 3107 21492 12503 21929 7921 142914398 22189 747 6439 12993 12307 19714 6650 18994 5932 18630 6683 192115651 5594 6958 13597 19763 10097 4065 14124 14687 1094 5780 7770 768815110 5797 7907 21169 3329 12627 19882 22067 19211 2061 7038 8909 1691413129 19881 16317 1551 13235 14888 1201 20264 7800 7526 2209 5887 1022310798 15317 11351 10105 10661 15014 2908 16412 22277 19851 12003 1961611003 7768 6166 4620 16231 13850 7016 2717 20886 14522 5254 15519 205413458 1240 15787 20099 9282 11480 4994 6281 18662 7992 9661 19875 111563619 14086 20948 16946 3456 6143 786 6709 5954 17926 20839 22351 1327715113 6368 7704 18694 3644 12154 3794 21257 21638 18386 18111 2149810731 13776 5539 14530 20282 7762 9529 17675 15191 12380 14865 158252818 16442 5901 8220 18578 13297 2495 21913 4526 16085 10965 15558 189822984 11245 21040 477 11140 2132 20312 4655 16568 21093 13882 5730 79767079 13035 2800 15563 509 12545 2037 11704 9571 21379 3712 7685 448318456 22341 11197 13107 13802 16006 428 534: 6383 10450 1006 8266 1798613269 9010 9822 18387 3735 10217 533 535 1774 16191 18891 13849 14706571 8356 3340 18461 20284 20753 10192 13836 5240 2653 20833 3564 187011250 6367 19001 20137 15672 13176 20207 8314 4015 1887 8795 21466 702318171 6876 8560 16477 13630 10793 4136 5249 17099 9152 2730 4724 86845821 3710 16562 22562 9500 21952 12129 1151 5863 2635 13579 5947 95436219 4851 7812 10216 19130 12258 1950 8843 1084 2519 12495 7141 18456128 9067 2618 1308 1202 16045 2887 3970 22176 2992 14189 2020 368718669 17082 7279 10946 10754 10583 4481 915 16903 18701 14442 1118212710 8108 4737 8174 21990 14565 20445 20022 9861 2888 17080 6216 34419629 4034 4019 17401 1207 22219 1481 5143 2857 17664 21589 11961 1915412425 21437 17489 11195 20933 21506 3533 12843 4677 18511 4730 696 956917797 18687 7097 20259 20966 15252 13221 11335 20777 12713 22502 1491521157 13596 7735 6111 14062 20371 3563 12591 16308 7626 21483 4212 1979819458 5691 18292 16130 21296 2832 830 14553 4632 6434 18295 17956 82912043 1189 3932 18731 6845 21360 6772 19039 21673 20779 1281 16128 1456817630 2371 4694 12396 15369 14260 13396 21621 8841 16662 11120 140816657 8692 9484 14065 2540 15204 6565 14320 2978 9294 7482 9493 1277 49527813 7306 12296 12021 6645 9286 22489 19908 893 14762 18330 10802 1047916663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060 6112 2245721974 20476 7333 6482 14526 7151 2644 835 10655 12264 9315 2786 162539488 5634 17372 2081 12643 15099 7425 15838 10094 788 3348 9280 2209518903 3706 15256 18593 5764 11115 12583 5630 11568 13613 2331 5136 807315998 11304 19137 5817 4430 5580 8588 18341 12540 2454 4970 17445 240110889 5190 13207 16465 11291 16479 21467 4263 2705 9673 20541 3458 124015787 20099 9282 11480 4994 2798 5475 1814 2683 8435 4345 3627 158979366 6281 20716 18662 7992 9661 19875 11156 21818 3619 14086 20948 169463456 6143 786 6709 5954 17926 20839 13891 10965 15558 15276 3513 1760011426 1123 16786 5326 17407 18982 2984 19817 11245 5838 21039 15310 213220312 4655 16568 21093 13882 5730 7976 7079 13035 2800 16353 4982 220323731 7388 13436 8698 509 12545 2037 11704 9571 21379 3712 7685 448318456 22341 15886 11197 13107 5444 16006 9573 20805 372 19913 9499 180577973 22459 21704 8464 535: 9382 1006 429 10217 2438 9883 314 18461 52406367 19001 16674 12484 526 11896 7812 9819 2857 17664 21589 11961 1915421437 11195 20933 21506 3533 4677 18511 4730 696 9569 17797 18687 709720259 15252 13221 450 11335 20777 12713 22502 14915 21157 20371 356312591 16308 7626 4212 19798 19458 5691 18292 16130 21296 2832 830 145536434 18295 17956 829 476 12043 20779 1281 17745 16586 7955 12705 15449988 20859 1768 11129 6547 22518 5552 9854 11376 6669 5359 9132 109641879 22087 13137 17298 3333 13752 20301 19370 2425 6946 19538 1427315340 6427 17165 15603 12419 10809 8003 13658 14064 4221 7387 1328420981 2316 3116 8418 3386 6508 18217 3578 22249 992 14855 13442 347119949 5277 19885 2816 10917 9436 18538 18659 12816 12308 15943 251520364 14701 8193 17579 22076 8890 2649 9329 21073 8376 15177 10882 8595990 9814 16053 5209 3144 13848 22428 19076 5454 17056 14918 4088 112705837 9604 22455 19840 12234 1149 801 953 9960 21301 1656 3755 1227822119 21098 1005 10264 13037 11702 20954 5581 5063 1118 6454 20632 169619474 14911 9381 4111 13374 2932 3071 18551 22362 1431 12923 6509 1922914012 5372 12362 17380 20272 16391 13395 5132 901 9540 19228 14568 176302371 4694 12396 15369 16597 3697 10741 16309 4927 13396 21621 8841 1666211120 14081 8692 9484 15204 6565 14320 2978 9294 7482 9493 1277 49527813 7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 15219 1696914762 18330 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 107098542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835 1065512264 9315 2786 16253 9488 5634 17372 788 9280 22095 18903 3706 1525618593 5764 11115 12583 11568 13613 2331 5136 8073 15998 5630 11304 191375817 5580 8588 18341 12540 2454 4970 17445 2401 11869 6193 21516 108895190 13207 16465 9673 9771 11716 18575 9584 12794 21399 20485 9218 186913350 18263 4846 544 19667 9933 4140 1318 20418 11128 20105 16734 237615699 7061 4232 15357 14036 5339 7107 19030 7165 21370 12103 4848 1321122530 15360 12863 9975 6398 14067 16683 21170 1924 890 8155 15866 101317187 4332 1235 20330 12927 7088 5099 2302 12424 8303 17466 14322 113832282 15956 3414 12982 18548 15665 10961 21084 10824 18440 16819 373013940 18821 14864 1818 19607 7969 6546 16771 5441 8459 18266 5000 1574913014 14274 8444 4707 13097 15930 11872 2621 7158 6942 18502 5408 1083721928 13800 5188 19614 16117 4719 1833 13499 3107 21492 12503 21929 79211429 14398 22189 6439 12993 12307 19714 6650 18994 747 5932 18630 66831921 15651 5594 6958 13597 19763 10097 14124 14687 1094 5780 7770 768815110 5797 7907 21169 3329 19882 12627 4065 22067 19211 2061 7038 890916914 13129 19881 16317 1551 13235 14888 1201 20264 7800 7526 2209 588710798 15317 10223 11351 10105 10661 15014 2908 16412 22277 19851 1200319616 11003 7768 6166 4620 13850 16231 7016 2717 20541 3458 1240 1578720099 9282 11480 4994 6281 18662 7992 9661 19875 11156 3619 14086 2094816946 3456 6143 786 6709 5954 17926 20839 22351 13277 15113 6368 770418694 3644 12154 3794 21257 21638 18386 18111 21498 10731 13776 553914530 20282 7762 9529 17675 15191 12380 14865 15825 2818 16442 5901 822018578 13297 2495 21913 4526 16085 10965 15558 18982 2984 11245 16474 304477 427 5067 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 280020416 509 307 305 12545 2037 11704 9571 21379 3712 7685 4483 18456 2234111197 13107 13268 16006 428 536: 8250 5293 17519 10969 8775 16532 1028611811 5689 2871 680 20415 14104 16888 17043 10273 6777 22396 602 486612333 9909 22327 4344 9005 16476 2573 10616 19638 15657 8950 4378 26573364 21708 985 6316 8776 2238 13856 1099 10466 19656 6879 9313 1960412069 8122 21385 16087 20487 1424 14078 2427 4175 7418 9385 16892 158093499 16714 5768 6322 13627 19469 8252 19551 14976 12742 10499 11322 95973569 9336 20132 5283 22127 10381 3149 7067 13886 7478 3897 20869 1939813086 4191 3669 20800 6799 8453 17539 21956 5724 13878 16687 1797 537:12225 7991 19769 20707 6346 21228 18920 15677 22007 17216 1246 1299 34828428 4864 1869 18259 5076 18894 10621 1194 21241 4280 10372 9939 346122101 14388 9687 1934 3266 19426 6888 17781 1697 1251 15687 11467 114447248 4960 3598 18026 17460 7270 18737 20522 18581 16569 13403 4999 209233253 12847 6541 12065 13811 442 18246 10039 21176 20831 13532 1907710287 9635 12724 898 4347 11482 12691 8702 12055 8006 14571 19675 1654221229 14891 11459 15395 15872 9301 10051 6843 5509 11707 10865 1890812031 9844 20469 4041 4623 22346 1849 12095 14281 3840 16733 15322 2178617305 10611 1428 7631 22045 6676 5894 8015 21902 14896 17330 3090 151227759 6196 16678 475 20224 3032 21119 21396 1165 20759 22152 18157 13373062 12222 10847 14596 3679 21087 17626 3743 10092 21220 22333 4831 63402684 14394 17353 12317 2073 22147 10166 9664 11412 21185 7389 1186211526 9220 2983 5429 13258 10529 12270 6868 5672 2355 11711 9314 177136624 12386 17898 3480 10028 20275 1292 12254 17594 20642 4853 10383 45918186 16379 15333 9201 15765 17720 5294 9934 7376 4038 2297 1916 714416736 10304 3319 3762 20614 11341 10440 4734 9677 12289 20772 4271 1995422175 3020 19748 16083 8948 19737 22114 2955 21137 3274 1408 6780 108487124 14060 11449 17939 4794 16058 21761 10251 4961 19141 1661 9701 62235983 18048 10670 17196 1709 4904 12196 630 8403 8582 6184 18819 33809202 16022 17350 12703 15237 17792 4597 11159 1072 20441 15476 1561217341 12261 21044 4892 1108 1406 22046 18198 6967 20318 12469 22123 764510452 1264 712 14602 6109 14712 22157 7745 16695 11050 22271 21574 538:8633 21066 20486 15659 19893 20341 18625 7202 9210 15561 8243 1945210486 9599 20104 5911 17836 3744 3313 22371 13393 8083 14598 8878 412111696 16024 6949 9760 18073 21021 1691 3955 13639 14076 5677 2739 1086020215 14300 10639 13585 11393 3392 7331 2940 4631 3646 14037 5709 83967169 3234 9515 13640 21141 9848 13213 16854 10018 17683 4171 20607 150412557 19483 17558 19443 20760 13774 16065 5097 13747 713 8505 18788 18352817 5302 8167 10335 22171 13497 18845 11474 8093 22332 17185 18392 153877019 17613 13932 22281 9232 21810 19046 14214 6221 10362 21188 121172909 9179 16519 21842 14950 5628 5467 18729 7240 8434 11000 2806 172051863 9645 18886 6782 20838 7708 6070 7788 22286 19842 16891 9527 180087791 11889 5849 2006 3823 12758 17857 15410 11552 11047 5550 18384 90318870 12346 18758 1298 11723 11231 13187 11334 10943 11898 11754 44926688 19935 13184 13651 1487 20841 6884 6464 8803 16050 6362 1430 136310992 6297 3031 2620 18570 22021 21438 13674 22415 1440 6599 17348 164032064 5725 621 12956 13371 3689 813 8410 17547 5880 7069 7272 13965 929618273 16456 7801 2112 17834 8758 7378 19838 6388 10579 19671 16279 80614835 2530 4277 795 12076 5622 13454 3935 4559 18529 18155 19209 156465247 6539 16369 5499 21300 14158 12126 17163 16184 993 21342 16623 1717210687 11363 1456 21186 21282 21603 10623 6475 17459 18745 21602 99963042 16551 1505 5224 5300 3833 4119 10925 11838 21509 15409 13868 1175316835 13881 7206 18880 6510 4201 3942 968 5155 13073 10765 5458 126733180 3108 15770 908 21482 5413 18381 14279 4351 15582 22158 16937 1241213635 14711 5969 9888 21962 11801 16194 10513 4703 11781 15593 1738318258 9184 7699 9109 1971 18780 3171 11419 10405 631 15583 21710 74835744 22254 12810 13894 20089 6909 5948 3120 15669 17412 19244 2016 1685022002 7260 14202 3293 4461 2271 14582 18598 17872 1569 18470 5671 89654082 7327 22511 15167 1160 15734 5450 19432 14387 14587 3176 8359 1816011357 15085 6948 5337 2567 10321 22182 10881 10168 18477 12537 1901520260 2170 18422 2440 3225 5484 1260 1822 7532 19248 7693 4353 104412660 5734 10038 19976 3453 19019 1674 12312 16581 10232 3727 1523 214022432 20580 9947 19324 20180 1384 2325 18901 10198 20110 9744 9208 318214144 18875 14329 929 22054 2752 4524 986 977 21117 9567 9679 1235220957 4772 12912 19652 11510 5608 21827 13765 8183 14331 13670 2050312206 21116 22320 844 2226 21795 13733 11051 20995 2615 7715 4478 229012549 15082 2491 2301 14182 15076 1227 10071 3792 13186 4722 1595 1088011269 8528 3213 14537 4257 8761 17108 4834 2651 19705 3355 7552 6681

Example 3 Consensus Sequence Build

ClustalW program was selected for multiple sequence alignments of theamino acid sequence of SEQ ID NO: 379 and 10 homologs. Three majorfactors affecting the sequence alignments dramatically are (1) proteinweight matrices; (2) gap open penalty; (3) gap extension penalty.Protein weight matrices available for ClustalW program include Blosum,Pam and Gonnet series. Those parameters with gap open penalty and gapextension penalty were extensively tested. On the basis of the testresults, Blosum weight matrix, gap open penalty of 10 and gap extensionpenalty of 1 were chosen for multiple sequence alignment. Attached arethe sequences of SEQ ID NO: 379, its homologs and the consensus sequenceSEQ ID NO: 22569 at the end. The symbols for consensus sequence are (1)uppercase letters for 100% identity in all positions of multiplesequence alignment output; (2) lowercase letters for >=70% identity;symbol; (3) “X” indicated <70% identity; (4) dashes “-” meaning thatgaps were in >=70% sequences.

SEQ ID NO  2406--------------------------MGSNGGSSNNNNNKVLEKPGQDQLVQQQQQQQE- 15414--------------------------MGSNGGSSNNNNNKVLEKPGQDQLVQQQQHPQE-   587------------------------------------MMGRVMEKPSQDLLQQQQQ-----  9696------------------------------------MMGRVMEKPSQDLLQQQQQ-----  5895------------------------------------MMGRVMEKPSQDLLQQQQQ----- 17251MFGNGNCDVDNEKTIITSSKWTQSEIDDHKVSMASSTGNRVMEKPGQELLQQQQQ----- 19549-----------------------------------------MEKQGQELLQQHHQQQQQQ   379-----------------------------------MQSKNMIVASSHQQQQQQQPQQPQP 21357---------MGLSSKQVSSSGLDWKQTLLEAQNLELPKPNLMRKQQQQQQQQQQQTONSE 17711--------MGLSSKQVSSSGLDWKQTLLEAQNLELPKPNLMRKQQQQQQQQQQQTQPNSE 13715-----------------------------------LTLTKCCMQRGSHFRSRSGSQEARS consensus--------------------------xxxxxxxxxxxxxxxxxxxxqxxxqqqqxxxxxx 22569APKCPRCDSSNTKFCYYNNYSLSOPRHECKACKRYWTRGGTLRNVPVGCCCRRNKRVKRP APKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRRNKRVERP ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP ALRCPRCDSSNTKFCYYNNYSLTQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRLKRP ALKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGYRRNNKRSTS QLKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGSYRKNKRVKRP SLKCPRCDSTNTKFCYYNNYNKSQPRHFCRACKRHWTKGGTLRNVPVGG-GRKNKRVKKS SLKCPRCDSTNTKFCYYNNYNKSQPRHFCRACKRHWTKGGTLRNVPVGG-GRKNKRVRKS GSSMSRCNSMDTKFCYYNNYNVNQPRHFCKNCQRYWTAGGSMRNVPVGAGRRKNKHTGSV xlkcpRCxSsnTKFCYYNNYslsQPRHFCkaCkRyWTrGGtlRNVPVGggxRkNkrvxxx LITTNPSSAAIDTAASNNSSN-SSSAPLQPPIDTASTS--------------NHINPLFY ITSPCSAAIDTASNSSNSSSAPTAAASLQPQIDTASTS--------------NHINPLFY TNHGDSSSSAANSPSSSNSNPPSQPHLDNIIASSSTTN------------HINNISPFFY TNHGDSSSSAANSPSSSNSNPPSQPHIDNIIASSSTTN------------HINNISPFFY TNHGDSSSSAANSPSSSNSNPPSQPHIDNIIASSSTTN------------HINNISPFFY TYPCSNNNNIDFSASPSSSTPSSVVANPNPPSQSQQQQQQQQHHSFDIAATSNHINTMLY SSNGPTSTTTLIKRPISTIETATTSNSSSPSSTHSSTS--------------NHMNPMFY STATTTTASTVSTTNSSSPNNPHQISHFSSMN----------------------HHPLFY ITPITTSSTTTTPITTATSTCTATVTTSIGNNNNNMDAMLG----------CYSHMTIQT ITTPITTSSTTTHQSQPPLQLALPQSQPQLATTTTTWMLCW----------VVIAT---- YRHTVITRDSLASLQVDGPDLVDHKPLSPEKVNGTILKEG--------------PDAPLC xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx----------xxxxxxxxxy GLPSSS-SDVNLPLFSRFGSRISSS----GEDLQLNNALGLGESSGVLSNEASDNNGYR- GLPSSS-SDVNLPLFSRFGSRISSS----GFDLQLNNALGLGESSRVLSNEASDNNRYR- GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN-- GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN-- GG-----DVMSSVPFPRFNLHSQLN------------ALGLGESTGVSENGESTSNNN-- GGNSCH-DVMNFPFSTRFNSTTRVSNPASGYDNLPQNGLGLGFSSGILMSAAGGEVNLNH GLSSTNNPCDPNLPFSRFNITSRLSTSSGYDLQPQMNFFGLGFSSGFENNGYTNGFNTS- GLSDHMSSCNNNLPMIPSRFSDSSK-----------TCSSSGLESEFLSSGFSSLSALG- PLADDQKNMSSSLYQALIRPPPLLLQQQNLLNTRELEGKDFGIGIGNGNNGIFPSSTLAL ESMASILNLGEQNLSSQLDFTAGAE-----------NREETSCSSACKPVKKKDITQHN- gxxxxxxxxxxxxxxxxxxxxxxxx----xxxxxxxxxxxxgxsxxxxxxxxxxxxxxx- -------------NWFGSNNTLLSSYTSTTSTTTPAMSSLLSSSLLQQKFMTDGVD---- SG-------------FGSNNMLLSSYTSTT-TTTPAMSSLLLQQKFISGGLKNDAD---- ------------SFFSAYNSMFGSSSSSTCAPSTPVMASLLSSTLLQQNLMGGGG---GG ------------SFFSAYNSMFGSSSSSTCAPSTPVMASLLSCTLLQQNFMGGGG---GG ------------SFFSAYNSMFGSSSSSTCAPSTPVMASLLSSTLLQQKLMSGGG---GG HHHHHHDEGSYRNGFSTSNNNNYSSIFGSSSTTTPVMASLLSSTLLQQKFMGTGGGIKGG ----------------NNNYDSIFSSSTSASNNTSVMPSVLSSTLLQHKFFDDGLK---- --------LGLPHQMSHDHTINGSFINNSTTNKPFLLSGLFGSSMSSSSTLLQHP-----P-------------IPHQSQSLLFPFSASSRSFDTNPCSVVSTSLRSSNVYNYGED---- ------------------------------------------------------------------------------------------------------------------------------------xxxxxxxxxxxxxxxxxxxxxxxxxxsxxxxxxxxxxxxxxxxx--xx --------STNTFQHGLGLTPLEQLQMASDHSSEAGMVALKDVKVELGQNNRLEWNNGAA --------SSNTFQHGLSLTSLEQLQIASDHSSEAGMVALKDVKVELGQNNNRLEWNGGA VKGRDHDQGDNTFHGLAPLQGLRVEGDSNNNIGSKEVKGEGQNRFEWSNNNNNNNNNGGG VKGRDHDQGDNTFHGLAPLQGLRVEGDSNNNIGSKEVKGEGQNRFEWSNNNNNNNNNGGG EGEEVVIMIKVATLSMAWHRYKGCKWKGIIIIVIILAQKK--------------------GGGGGGDDDPFHHHQEMDSKEVKLGEGLQNRLDQWNMNNLNGNGGAVFQNQMENMGLSDN ----------YGSDAGSNGAFQDLQFGSKMQNQMEHIGGFYDPASSIYLNATSSSAIGVW ----------HKPMNNGGDMLGQSHLQTLASLQDLHVGGNNEDMKYKEGKLDQISGNING ---------QFKAIEEPTINSTTATIVPSTGGTNNTHHPWEIAAATSGVGLGTSSNSNYW xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx CQSQIQHVGLYDPSLYWNNSAATALGVWNDQAANIGSSVTSLI FQSQIQHVGLYDPLLYWNN-SATALGVWNDQAANIGSSVTSLI GQNQMEHVGLSDPNSLYWN-TATGLGAWSDQPNNIGPSVTSLI GQNQMEHVGLSDPNSLYWN-TATGLGAWSDQPN-IGPSVTSLI -------------------------------------------NASLYWNNNHNNSNNNTSA-TATGLSSVWSTDQPGSNSVSSLI NDQGANNIGSSVTSLI --------------------------FMSSSSSLDPSNYNNMWNNASVVNGAWLDPTNNNVGSSLTSLI NWEDEDSLVSTDLKDPWDDSDIKP---------------------------------------------------------------------------------------------------------xxxxxxxxxxxxxxxxxxx-xxxxxxxxxxxxxxxxxxxxxxx 

Example 4 Corn Transformation Construct

GATEWAY™ destination vectors (available from Invitrogen LifeTechnologies, Carlsbad, Calif.) are constructed for insertion oftrait-improving DNA for corn transformation. The elements of eachdestination vector are summarized in Table 17 below and include aselectable marker transcription region and a DNA insertion transcriptionregion. The selectable marker transcription region comprises aCauliflower Mosaic Virus 35S promoter operably linked to a gene encodingneomycin phosphotransferase II (nptII) followed by both the 3′ region ofthe Agrobacterium tumefaciens nopaline synthase gene (nos) and the 3′region of the potato proteinase inhibitor II (pinII) gene. The DNAinsertion transcription region comprises a rice actin 1 promoter, a riceactin 1 exon 1 intron1 enhancer, an att-flanked insertion site and the3′ region of the potato pinII gene. Following standard proceduresprovided by Invitrogen the att-flanked insertion region is replaced byrecombination with trait-improving DNA, in a sense orientation forexpression of a trait-improving protein and in a gene suppressionorientation (i.e., either anti-sense orientation or in a sense- andanti-sense orientation) for a trait-improving suppression of a protein.Although the vector with trait-improving DNA inserted at the att-flankedinsertion region is useful for plant transformation by direct DNAdelivery, such as microprojectile bombardment, it is preferable tobombard target plant tissue with tandem transcription units that havebeen cut from the vector. For Agrobacterium-mediated transformation ofplants the vector also comprises T-DNA borders from Agrobacteriumflanking the transcription units.

Vectors for Agrobacterium-mediated transformation are prepared with eachof the trait-improving genes having a sequence of SEQ ID NO:1 throughSEQ ID NO:269 with the DNA solely in sense orientation for expression ofthe encoded, cognate trait-improving protein and in a gene suppressionorientation for suppression of the cognate protein. Each vector istransformed into corn callus which is propagated into a plant that isgrown to produce transgenic seed for each transgenic event. Progenyplants are self-pollinated to produce seed which is selected forhomozygous seed. Homozygous seed is used for producing inbred plants,for introgressing the trait into elite lines, and for crossing to makehybrid seed. The progeny transgenic plants comprising thetrait-improving DNA with a sequence of SEQ ID NO: 1 through SEQ ID NO:269 have one or more improved traits identified by agronomic traitscreening including, but not limited to, enhanced nitrogen useefficiency, increased yield, enhanced water use efficiency, growth undercold stress and enhanced oil, starch and protein levels. Transgenic cornincluding inbred and hybrids are also produced with DNA from each of theidentified homologs of DNA of SEQ ID NO: 1 through SEQ ID NO: 269 toprovide transgenic seeds and plants which are identified from totaltransgenic events by screening for the improved agronomic trait.Transgenic corn plants are also produced where the trait-improving DNAis transcribed by each of the promoters from the group selected from, amaize globulin 1 promoter, a maize oleosin promoter, a glutelin 1promoter, an aldolase promoter, a zein Z27 promoter, a pyruvateorthophosphate dikinase (PPDK) promoter, a soybean 7S alpha promoter, aperoxiredoxin antioxidant (Per1) promoter and a CaMV 35S promoter.

Seed produced by the plants is provided to growers to enable productionof corn crops with improved traits associated with the trait-improvingDNA.

TABLE 17 FUNCTION ELEMENT REFERENCE DNA insertion Rice actin 1 promoterU.S. Pat. No. 5,641,876 transcription region Rice actin 1 exon 1, intron1 U.S. Pat. No. 5,641,876 enhancer DNA insertion AttR1 GATEWAY ™CloningTechnology transcription region Instruction Manual (att-flanked insertinCmR gene GATEWAY ™Cloning Technology region) Instruction Manual ccdA,ccdB genes GATEWAY ™Cloning Technology Instruction Manual attR2GATEWAY ™Cloning Technology Instruction Manual DNA insertion PotatopinII 3′ region An et al., (1989) Plant Cell 1: 115-122 transcriptionregion selectable marker CaMV 35S promoter U.S. Pat. No. 5,858,742transcription region nptII selectable marker U.S. Pat. No. 5,858,742 nos3region U.S. Pat. No. 5,858,742 PinII 3′ region An et al., (1989) PlantCell 1: 115-122 E. coli maintenance ColE1 origin of replication regionF1 origin of replication Bla ampicillin resistance

Example 5 Soybean Transformation Construct

Constructs for use in transformation of soybean are prepared byrestriction enzyme based cloning into a common expression vector.Elements of an exemplary common expression vector are shown in Table 18below and include a selectable marker expression cassette and a gene ofinterest expression cassette. The selectable marker expression cassettecomprises Arabidopsis act 7 gene (AtAct7) promoter with intron and5′UTR, the transit peptide of Arabidopsis EPSPS, the synthetic CP4coding region with dicot preferred codon usage and a 3′ UTR of thenopaline synthase gene. The gene of interest expression cassettecomprises a Cauliflower Mosaic Virus 35S promoter operably linked to atrait-improving gene in a sense orientation for expression of atrait-improving protein and in a gene suppression orientation (i.e.,either anti-sense orientation or in a sense- and anti-sense orientationfor a trait-improving suppression of a protein.

Vectors similar to that described above are constructed for use inAgrobacterium mediated soybean transformation systems, with each of thetrait-improving DNA having a sequence of SEQ ID NO:1 though SEQ IDNO:269 and the respective identified homologs with the DNA in senseorientation for expression of the encoded, cognate protein and in a genesuppression arrangement for suppression of the cognate protein. Eachvector is transformed into soybean embryo tissue to produce transgenicevents which are grown into plants that produce progeny transgenicplants and seed for screening to identify the transgenic soybean plantsof this invention that exhibit the enhanced agronomic trait imparted byDNA with a sequence of SEQ ID NO:1 through SEQ ID NO:269 or a respectivehomolog. The transgenic soybean plants of this invention are identifiedby agronomic trait screening including, but not limited to, enhancednitrogen use efficiency, increased yield, enhanced water use efficiency,growth under cold stress and enhanced oil, starch and protein levels.Transgenic soybean plants are also produced where the trait-improvingDNA is transcribed by a napin promoter and Arabidopsis SSU promoter.

Seed produced by the plants is provided to growers to enable productionof soybean crops with improved traits associated with thetrait-improving DNA.

TABLE 18 Function Element Reference Agro transformation B-ARGtu.rightborder Depicker, A. et al (1982) Mol Appl Genet 1: 561-573 Antibioticresistance CR-Ec.aadA-SPC/STR Repressor of primers from the ColE1plasmid CR-Ec.rop Origin of replication OR-Ec.oriV-RK2 Agrotransformation B-ARGtu.left border Barker, R. F. et al (1983) Plant MolBiol 2: 335-350 Plant selectable marker expression cassette Arabidopsisact 7 gene McDowell et al., (1996) Plant (AtAct7) promoter with Physiol.111: 699-711. intron and 5′UTR 5′ UTR of Arabidopsis act 7 gene Intronin 5′UTR of AtAct7 Transit peptide region of Klee, H. J. et al (1987)MGG Arabidopsis EPSPS 210: 437-442 Synthetic CP4 coding region withdicot preferred codon usage A 3′ UTR of the nopaline synthase U.S. Pat.No. 5,858,742 gene of Agrobacterium tumefaciens Ti plasmid Plant gene ofinterest expression cassette Promoter for 35S RNA from U.S. Pat. No.5,322,938 CaMV containing a duplication of the −90 to −350 region Geneof interest insertion site Cotton E6 3′ end GenBank accession U30508

Example 6 Cotton Transformation

Vectors similar to that described above for soybean transformation areconstructed for use in Agrobacterium mediated cotton transformationsystems, with each of the trait-improving DNA having a sequence of SEQID NO:1 though SEQ ID NO:269 and the respective identified homologs withthe DNA in sense orientation for expression of the encoded, cognateprotein and in a gene suppression arrangement for suppression of thecognate protein. Each vector is transformed into cotton embryo tissue toproduce transgenic events which are grown into plants that produceprogeny transgenic plants and seed for screening to identify thetransgenic soybean plants of this invention that exhibit the enhancedagronomic trait imparted by DNA with a sequence of SEQ ID NO:1 throughSEQ ID NO:269 or a respective homolog. The transgenic cotton plants ofthis invention are identified by agronomic trait screening including,but not limited to, enhanced nitrogen use efficiency, increased yield,enhanced water use efficiency, growth under cold stress and enhancedoil, starch and protein levels. Transgenic cotton plants are alsoproduced where the trait-improving DNA is transcribed by a napinpromoter and Arabidopsis SSU promoter.

Seed produced by the plants is provided to growers to enable productionof cotton crops with improved traits associated with the trait-improvingDNA.

We claim:
 1. A method of producing a crop comprising growing transgenicseed for a crop, wherein the genome of said transgenic seed comprises arecombinant DNA comprising a nucleotide sequence encoding a proteinhaving an amino acid sequence with at least 99% amino acid sequenceidentity to the amino acid sequence of SEQ ID NO: 505 and having thefunction of the protein of SEQ ID NO: 505, thereby producing apopulation of transgenic plants, and selecting from the populationtransgenic plants a transgenic plant which expresses said proteinencoded by said nucleotide sequence and having an enhanced trait ascompared to a control plant of the same plant species lacking saidrecombinant DNA in their nuclei, wherein said enhanced trait is selectedfrom the group of enhanced traits consisting of enhanced cold tolerance,enhanced heat tolerance, enhanced resistance to salt exposure, enhancedyield and enhanced nitrogen use efficiency.
 2. The method according toclaim 1, wherein transgenic plants grown from said transgenic seedexhibit increased yield as compared to a control plant of the samespecies lacking said recombinant DNA.
 3. The method according to claim1, wherein said selected transgenic plant exhibits increased yield ascompared to a control plant of the same species lacking said recombinantDNA when said selected transgenic plant is grown in a yield-limitingenvironment of low nitrogen availability.
 4. The method according toclaim 1, wherein said selected transgenic plant exhibits increased yieldas compared to a control plant of the same species lacking saidrecombinant DNA when said selected transgenic plant is grown in ayield-limiting environment of water deficit stress.
 5. The method ofclaim 2, wherein the protein has the amino acid sequence of SEQ ID NO:505.
 6. The method according to claim 1, wherein said selectedtransgenic plant exhibits increased yield as compared to a control plantof the same species lacking said recombinant DNA when said selectedtransgenic plant is grown in a yield-limiting environment of highsalinity stress.
 7. The method according to claim 1, wherein saidselected transgenic plant exhibits increased yield as compared to acontrol plant of the same species lacking said recombinant DNA when saidselected transgenic plant is grown in a yield-limiting environment ofcold stress.